RNA APTAMERS SPECIFIC FOR THE HCV NS3 PROTEASE AND HELICASEDOMAINS
Kotaro Fukuda1, 2*, Fumiko Nishikawa2, Takuya Umehara1, 2, Joonsung Hwang2, Nobuko,
Kakiuchi2, Satoshi Nishikawa2 and Tsunemi Hasegawa1
1
Department of Material and Biological Chemistry, Faculty of Science, Yamagata University,
Yamagata 990-8560, Japan
2
National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki
305-8566, Japan
Present address:
Takuya Umehara: Department of Molecular Biophysics and Biochemistry, Yale University, 266
Whitney Avenue, New Haven, CT 06520, USA
Joonsung Hwang: Developmental Skin Biology Unit, NIH/NIAMS, Bethesda MD 20892, USA
Nobuko Kakiuchi: Graduate School of Natural Science and Technology, Department ofPharmaceutical Chemistry, Kanazawa University, Kakuma, Kanazawa 920-1192, JAPAN*
Corresponding author:
Department of Material and Biological Chemistry, Faculty of Science, Yamagata University,
Yamagata 990-8560, Japan. FAX: 023-628-4604; E-mail: kotaro.f@sci.kj.yamagata-u.ac.jp(Received October 16, 2006 Accepted October 25, 2006)
Abstract
The hepatitis C virus (HCV)
non-structural protein 3 (NS3) is a multifunctional
enzyme with protease and helicase activities. It isessential for HCV replication and proliferationand is therefore a target for anti-HCV drugs. To
obtain efficient RNA aptamers specific for HCV
NS3, and to develop inhibitors of HCV
replication, we performed in vitro selection for
the NS3 protease and helicase domains,respectively. In vitro selection, namely, SELEX(systematic evolution of ligands by exponential
enrichment) is a useful strategy for isolating
nucleic acid sequences that have a high affinity
for a target molecule from a randomized
oligonucleotide pool. Isolated RNA aptamers
showed effective inhibition against either theprotease or helicase activity of NS3 in vitro. In
addition, new type of RNA ligands, whichdisplayed dual-inhibitory functions targeting both
NS3 protease and helicase activities wereconstructed. Furthermore, NS3 protease aptamers
effectively inhibited NS3 protease activity inliving cells.
Keywords: HCV, NS3, protease, helicase, in
vitro selection, SELEX, aptamer
Introduction
The hepatitis C virus (HCV) is the
major etiological agent of non-A, non-B hepatitis,
with a worldwide carriage rate of 3%. Most
patients develop chronic hepatitis, and persistentinfection often leads to liver cirrhosis or
hepatocellular carcinoma. Recently, the mosteffective treatment for HCV has been found to be
a combination of interferon and ribavirin.
However, not all subtypes respond to treatment
and the drugs can cause serious side effects [1].
The development of anti-HCV drugs with greater

safety and efficacy is therefore a priority.
HCV is a single-stranded RNA virusthat belongs to the Flaviviridae family [2]. The~9.6-kb genome of positive polarity encodes aprecursor polyprotein (~3,010 amino acids),
which is processed to structural (core protein C,
and envelope glycoproteins E1 and E2) andnon-structural (NS2, NS3, NS4A, NS4B, NS5A
and NS5B) proteins by a host signal peptidase andtwo viral proteases, NS2-3 and NS3 [3]. The NS3protein is a multi-functional enzyme with atrypsin-likeprotease
withintheamino(N)-terminal one-third, and an NTP-dependent
RNA/DNA helicase in the remaining two-thirds[4–6]. It is generally accepted that the proteasedomain cleaves the junctions between the
non-structural proteins, together with cofactor
NS4A [7]. The helicase domain unwinds the
double-stranded RNA generated by the
RNA-dependent RNA polymerase NS5B during
genome replication [8, 9]. As NS3 is essential for
HCV replication and proliferation, new anti-HCV
drugs could potentially be directed against thisprotein [10, 11]. One source of potential highly
selective NS3 inhibitors might be RNA ligands,
called RNA aptamers. Several RNA aptamers
specific for HCV NS3 have been identified using
an in vitro selection method (namely, systematic
evolution of ligands by exponential enrichment[SELEX]) in our laboratory [12–16].
SELEX is a protocol for isolating rare
nucleic acid sequences that have high affinity fora target molecule from a randomized
oligonucleotide pool [17, 18]. In vitro selection
has been used to isolate high affinity DNA or
RNA ligands for target molecules including
proteins, organic dyes, and amino acids [19]. Like
antibodies, aptamers fold on the basis of nucleic
acid sequence into three-dimensional structuresthat bind to their targets. Thus, aptamer binding to
its target is a highly specific interaction,

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discriminating between related proteins that sharecommon sets of structural domains. Therefore,
aptamers are an attractive class of molecules foruse as therapeutic compounds. This review
describes the isolation and characterization of
RNA aptamers that bind with high specificity to
the HCV NS3 protease and helicase domains,respectively. Furthermore, application of NS3
protease aptamers in vivo as antiviral compounds
against HCV is also evaluated.
RNA aptamers specific for the HCV NS3protease domain
Since the NS3 protease of HCV isessential for processing the viral polyprotein, its
inhibition
blocksviralreplication
andproliferation. Thus, there much interest in thepossibility of using an inhibitor of the NS3protease as an anti-HCV drug. To inhibit the HCV
NS3 protease activity, RNA aptamers specific for
the NS3 protease domain (∆NS3) were selectedby in vitro selection using a synthetic 30N
random RNA pool [12]. Highly efficient RNA
aptamers G9-I, -II, and -III (Table 1) wereselected that contain a conserved loop sequence[5’- GA(A/U)UGGGAC -3’] and were efficientinhibitors of the NS3 protease in vitro. The NS3protease activity toward a synthetic substrate wasalmost completely inhibited by a fivefold molar
excess of the selected RNA aptamer (Fig. 1A and
1B). Furthermore, even in the presence of P41
peptide from NS4A, which is a stimulating
cofactor for NS3 protease, 70% of the proteaseactivity was inhibited by the selected RNA
aptamers (Fig. 1C). In addition, these aptamers
are all specific to ∆NS3 and do not bind to any
other member of trypsin-like serine proteases,
such as trypsin, chymotrypsin, α-thrombin, andactivated protein C. In the case of the G9-I
aptamer, both Kd and Ki values were in the nM
range and mutational analysis demonstrated thatthe conserved loop sequence is crucial for theprotease inhibition.

Fig. 1. Inhibition of the NS3 protease with RNA
aptamers. The substrate S1 (86 µM) was mixed
with (A) ∆NS3 (0.75 µM) and RNA aptamer (3.6µM), (B) MBP-NS3 (2.0 µM) and RNA aptamer(10.0 µM) or (C) MBP-NS3 (2.3 µM), RNAaptamer (11.3 µM) and NS4A peptide (P41; 22.5µM). After incubation, the reaction mixture wasanalyzed on a reverse phase HPLC and relative
protease activity was calculated by normalizing to
the activity in the reaction containing only ∆NS3 or
MBP-NS3 (C, control).

The RNA aptamer-binding site of HCV NS3protease
Further kinetic analysis of G9-I
aptamer (an NS3 protease aptamer) indicated that
G9-I aptamer is noncompetitive (i.e., does notcompete with the substrate at the catalytic pocketof the protease) [12]. To elucidate the binding siteof G9-I aptamer in NS3 protease domain, we
carried out alanine scanning mutagenesis at
positive charged residues on the surface of ∆NS3[20]. The result of binding analysis by surface
plasmon resonance measurements and proteaseinhibition assay clarified that Arg161 as well as-134-

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Arg130 of ∆NS3 are essential for interaction with
G9-I aptamer.
It is speculated that Arg161 and Lys165
in NS3 protease are located near the P6 position,
which is the site of the conserved acidic aminoacid that binds to the substrate [21]. X-ray
structural analysis of entire NS3 protein [22]suggests that both local and global conformationalchanges occur during processing of HCV
polyprotein. The polyprotein substrate itself may
promote structural changes by forming a weaksolvent-exposed salt linkage between the P6
acidic residue of the substrate and the basic(positive) amino acids, Arg161 and Lys165, inNS3. These residues may play a critical role insubstrate recognition. Thus, this region appears to
be a potential targeting site for anti-HCV drugs.
RNA aptamers specific for the HCV NS3
helicase domain
To obtain efficient RNA aptamers
specific for the helicase domain of HCV NS3, andto develop an inhibitor of replication of HCV
mRNA, we performed in vitro selection for theNS3 helicase domain using an RNA library
containing N30 random core sequences [13].
Aptamers were obtained after eight rounds of
selection using the competitor (poly U), which
inhibited the helicase activity of NS3. Most RNA
aptamers possessed 5’ extended single-stranded

regionsandtheconserved
sequence[5’-GGA(U/C)GGAGCC-3’] in the stem-loop
regions. Among them, aptamer #5 (Table 1)
showed the highest inhibition activity (IC50 ≈ 50nM) against full-length NS3. Deletion andmutagenesis analysis of aptamer #5 showed thatthe conserved stem-loop is important and that thewhole structure is needed for helicase inhibition.
Characterization of aptamer #5 led tothe proposed mechanism of HCV replication.
During the virus life cycle, the 3’(+/-)UTR of
HCV is essential for replication and facilitatesformation of the RNA polymerase complex
containing non-structural viral proteins. NS3 hasstrong affinity for HCV 3’(+)UTR whose
secondary structure is composed of three uniquedomains: a variable region (30-40 nt) and a poly(U) or poly (U/UC) tract (20-200 nt) followed by
the highly conserved 3’X region [23]. There issomeconfigurational
similarity
between3’(+)UTR and RNA aptamers against NS3
helicase but no sequence similarity. Comparisonof the helicase inhibition activity of aptamer #5
and HCV 3’(+)UTR led to the speculation that thevariable region in 3’(+)UTR may regulate theNS3 helicase activity. Considering the expected
involvement of this region in the regulation ofNS3, the poly (U/UC) tract might provide anassembly site for NS3 to form the replication
complex with other proteins. Therefore theTable 1. The secondary structures of NS3 protease and helicase aptamers.

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strength of helicase inhibition might be affectedby the efficiency of replication.
An RNA ligand inhibits HCV NS3 proteaseand helicase activities
NS3 helicase has been reported to bind
preferentially to the poly(U) sequence, which islocated in the 3′ untranslated region (UTR) of the
HCV genome [23] and is necessary for viral
replication [24]. The substrate-binding site is
located along the cleft between the first two
domains of the helicase and the third domain of
the tertiary structure, complexed with adeoxyuridine octamer (dU8) [25]. As mentioned
above, we obtained RNA aptamers thatspecifically and efficiently inhibited NS3 proteaseactivity (G9 aptamers). To add helicase-inhibition
capability, we attached (U)14 to the 3′-terminalend of a minimized G9 aptamer, ∆NEO-III [14].
The resulting NEO-III-14U (Table 1) was shownto inhibit NS3 protease activity more efficiently
than the original aptamer and, furthermore, toefficiently inhibit the unwinding reaction by NS3

helicase. In addition, NEO-III-14U had thepotential to suppress specific interaction between
NS3 and the 3′ UTR of HCV-positive and-negative strands.
Members of the Flaviviridae family,such as the yellow fever virus, Dengue virus,
Japanese encephalitis virus, and bovine viraldiarrhea virus 1, are also known to have an NS3protein with both protease and helicase domains[26]. Thus, the new strategy of designing ananti-viral RNA inhibitor by fusing a specificprotease aptamer to a short single-stranded
oligonucleotide should be applicable to other viralNS3 proteins.
Rational design of dual-functional aptamersthat inhibit the protease and helicase activities
of HCV NS3
To develop a novel aptamer withenhanced dual-inhibitory activity against theprotease and helicase functions of NS3, advanced
dual-functional (ADD) aptamers were created byfusingprevious
aptamers
with
known

Fig. 2. Schematic diagram of an aptamer expression plasmid. The HDV-G9-II aptamer (HA) conjugated withCTE M45 (HAC) is designed to be transcribed under the control of the CAG promoter (Chicken ß-actin promoter+ cytomegalovirus enhancer, indicated by CAG). The arrow indicates the self-cleavage site of the cis-acting HDV
ribozyme. Protease inhibition activity of the tandem chimeric aptamer, (HAC)n, is related to the increase in thenumber (n) from 1 to 4 [16].

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anti-protease and anti-helicase activities [15]. The
structural domain of the helicase aptamer, #5∆,
was conjugated via an oligo(U) tract to the 3'-endof the dual functional aptamer NEO-III-14U or
the protease aptamer G9-II. The spacer length was
optimized to obtain two ADD aptamers,
NEO-35-s41 and G925-s50 (Table 1); both weremore effective inhibitors of NS3 protease/helicase
activity in vitro, especially against the helicase
activity with a four- to fivefold increase ininhibition compared to #5 and NEO-III-14U.
Furthermore, G925-s50 effectively inhibited NS3
protease activity during HCV replication in vitro.
Development of other aptamer conjugates will
increase the range of their biological application.
Inhibition of HCV NS3 protease by RNA
aptamers in cells
The inhibitory activity of aptamers
within living cells was investigated bytransfecting HeLa cells with various aptamers.
The HeLa cells transiently expressed NS3 and asubstrate protein (two types of GFP derivatives
linked by a NS3 cleavage site). Using this system,
FRET could easily detect inhibition of NS3
protease activity [27] and showed that
NEO-III-14U, NEO-35-s41, and G925-s50
inhibited around 50% of NS3 proteolysis in vivo
[14, 15].
Furthermore, we constructed the G9-II
aptamer expression system in cultured cells using
CAG promoter (cytomegarovirus enhancer +chicken ß-actin globin promoter) [16]. We
designed a chimeric HDV ribozyme-G9-II
aptamer (HA) conjugate, which was used to
produce stable RNA in vivo and tandem repeats ofthe functional unit (Fig. 2). In addition, tofacilitate the localization of transcribed RNA
aptamers in cytoplasm, the minimal mutant of
CTE (Constitutive Transport Element), derived
from the type D retrovirus, was conjugated at the
3'-end of HA (HAC) (Fig. 2). Transcript RNAs,(HA)n and (HAC)n, were processed into theproper units containing the G9-II aptamer by thecis-acting HDV ribozyme, which was confirmed
both in vitro and in vivo. The efficient proteaseinhibition activity of the HDV ribozyme-G9-II
aptamer expression plasmid was demonstrated in
HeLa cells. Protease inhibition activity of thetandem chimeric aptamer, (HA)n and (HAC)n,increased in relation to the number (n) from 1 to 4.
This tandem system involving HDV ribozyme
will be useful for controlling the expression offunctional RNAs in cultured cells.
These lines of research have thepotential to lead to the development of NS3
protease and helicase aptamers as anti-HCV
compounds.
Acknowledgements
We thank Dr. K. Shimotohno (Virus
Reseach Institute, Kyoto University) for thegenerous gift of NS3-4A expression plasmid. We
thank Dr. M. Kohara (Tokyo Metropolitan

Institute of Medical Science, Japan) for providing
in vitro HCV-replication system. We thank Dr. S.
Sekiya and Mr. K. Funaji (Mitsubishi Gas
Chemical Company, Inc., Japan) for helpful
discussions.
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