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Visible Sensing of Nucleic Acid Sequences with a Genetically Encodable Unmodified RNA Probe.

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DNA Sensing
DOI: 10.1002/ange.200503836
Visible Sensing of Nucleic Acid Sequences with a
Genetically Encodable Unmodified RNA
Atsushi Narita, Kazumasa Ogawa, Shinsuke Sando,*
and Yasuhiro Aoyama*
As the amount of identified genetic information continues to
grow, there has been much current interest in rapid and
simple gene sensing with high sequence selectivity.[1] Among
various types of homogeneous sensing methods,[1, 2] such as the
use of molecular beacons (MBs; Figure 1 a),[2a,b] several new
strategies have enabled catalytic sensing of a target sequence
over the equivalent stoichiometry of the target molecule
through a signal-amplifying process.[3–12] These strategies
enzyme-coupled[6, 7]
approaches, template-directed catalytic chemical reaction
probes,[8, 9] and a method with electrochemical catalysis[10] or
mechanistically controllable magnetic particles.[11] Recently,
we reported a new strategy with MB–mRNA systems for
sensitive genotyping.[12] The strategy was based on the system
of naturally occurring[13] or engineered[14] hairpin-shaped
RNAs for conformation-induced control of translation frequency, so that the sensing can be conducted by using
genetically encodable unmodified RNA as a probe in a typical
prokaryotic translation system.
The artificial translation regulation system is composed of
a cis-acting MB-like RNA structure, wherein the loop region
(green in Figure 1 a) is complementary to the target and the
stem is composed of sequences for a RBS (red) and the antiRBS or RBS-docking domain complementary thereto
(pink).[12] Target binding is designed to result in the opening
of the MB structure, thereby making the RBS domain
accessible by the ribosome and hence initiating the translation
of a reporter gene, such as luciferase. Although the MB–
mRNA system actually realized the sensitive detection of
nucleic acids through the double signal-amplifying process of
catalytic translation and catalytic signal transduction through
enzymatic reaction of the translated reporter protein, the
[*] A. Narita, K. Ogawa, Dr. S. Sando, Prof. Dr. Y. Aoyama
Department of Synthetic Chemistry and Biological Chemistry
Graduate School of Engineering
Kyoto University
Katsura, Nishikyo-ku, Kyoto 615-8510 (Japan)
Fax: (+ 81) 75-383-2767
[**] This work was supported by an Industrial Technology Research
Grant Program from the New Energy and Industrial Technology
Development Organization (NEDO) of Japan and also partly by
Grants-in-Aid (Grant nos.: 16350087 and 17034026) from the
Japanese Government. A.N. acknowledges a fellowship from JSPS.
Supporting information for this article is available on the WWW
under or from the author.
Angew. Chem. 2006, 118, 2945 –2949
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. a) Illustration of MB, MB–mRNA, and RNase H activity
coupled MB–mRNA systems. The ribosome-binding site (RBS), antiRBS or RBS-locking site, start codon, and target-binding domain are
colored red, pink, blue, and green, respectively. b) Predicted secondary
structures of the 5 ’-end region of MB–mRNA targeting the nucleotide 620–637 or 21–36 regions of the human CCR5 gene sequence.
c) Sequences of the 620–637 and 21–36 regions of the target human
CCR5 gene.
method still has two major shortcomings. The first is sequence
selectivity. The target-binding domain should be sufficiently
long to open the MB (hairpin) structure, so that the probe
fails to discriminate single nucleotide differences, for example, the C to T transition that forms a relatively stable GT
mismatch in the target/probe heteroduplex. The second is
sensitivity. As stoichiometric binding of the target to the
probe is required for continuous translation of a reporter
protein, the translation-activated mRNA probe is, at best,
equimolar to the target; this typically leads to low signal
intensity, especially when the target is present in a tiny
The present work is concerned with an RNase H activity
coupled MB–mRNA system. The RNase H activity coupled
approach allows triple catalytic sensing of target nucleic acids
with improved selectivity and enhanced sensitivity. In addition, we report herein an application for the visible sensing of
nucleic acids with an unmodified RNA probe in this RNase H
activity coupled system.
RNase H is an endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA in the DNA/RNA
heteroduplex.[15] Therefore, an MB–mRNA system coupled
with the RNase H activity could drive an additional process
for catalytic production of a translation-activated mRNA
probe, wherein the target-bound loop region (target = oligodeoxynucleotide (ODN)) is digested by coexisting RNase H
to release an anti-RBS stem domain away from the mRNA
body, thus allowing catalytic use of target DNA (Figure 1 a).
To demonstrate the designed triply catalytic gene sensing,
we first used an mRNA probe composed of the MB domain
targeting the nucleotide 620–637 site (18 nucleotides (nts)) of
the human CC chemokine receptor 5 (CCR5) gene sequence
(Figure 1 b),[16] downstream of which is a codon-optimized
luciferase reporter gene, derived from pBESTluc vector
(Promega). The sensing of target CCR5 ODNs (Figure 1 c)
with the MB–mRNA probe ( 1.8 pmol in 10 mL solution,
0.1 mg mL 1) was carried out at 37 8C for 1 h in a reconstituted
prokaryotic translation system (Pure System)[17, 18] in the
presence or absence of additional Tth RNase H (typically
0.01 U mL 1); this was followed by a chemiluminescence (CL)
assay of the luciferase expressed with a 96-well microplate
reader (Wallac 1420 system; Figure 2). In the absence of
RNase H, an equimolar amount of fully matched target
ODNfull afforded a moderately enhanced CL (Ion/Ioff = 314 %)
relative to that in the absence of the target (lane 1 versus
lane 2 in Figure 2 a). An RNase H activity coupled
(0.01 U mL 1) translation system, under otherwise identical
conditions, resulted in an enhanced translation activation that
reached 615 % CL enhancement (lane 3 versus lane 4 in
Figure 2 a).
Control runs indicated that RNase H indeed catalyzed the
target-assisted cleavage of the probe.[19, 20] The probe can also
be provided in the form of double-stranded (ds) DNA, from
which the MB–mRNA will be transcribed in situ. Under the
RNase H coupled T7-transcription/translation conditions, the
precursor dsDNA (0.2 pmol) gave a further enhanced value
of Ion/Ioff = 1320 % for an equimolar amount of target ODNfull
(lane 1 versus lane 2 in Figure 2 b). Nucleic acid sensing for a
different CCR5 sequence was further conducted by using a
newly designed MB-type cis-repressing domain targeting
nucleotides 21–36 of CCR5 (Figure 1 b). As seen in Figure 3,
the presence of a fully matched sequence afforded highly
enhanced CL under the RNase H activity coupled conditions,
with Ion/Ioff = 953 % (lane 2 versus lane 3 in Figure 3), thus
partly revealing the generality of the target sequence for this
triple catalytic sensing system.
The sensitivity-enhanced RNase H activity coupled
system still retained a low selectivity for single-nucleotide
differences in the 18-nt binding site (lanes 5–7 in Figure 2 a).
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. 2006, 118, 2945 –2949
One-nucleotide selectivity was further
investigated by using the shorter target of
16 nts (the nucleotide 620–635 region of
the CCR5 gene sequence in Figure 1 c).
The shorter target length could lead to
higher sequence selectivity; however, the
target/probe hybridization of 16 nts seems
to be too short to invade into the hairpin
domain (a total of 13 internal base pairings) and open the MB structure by the
presence of only one equivalent of a target
with no external support, as revealed by
the design of the conventional MB probe.
This was the case. As shown in Figure 2 c
(lanes 2 and 3), the original system that was
not RNase H activity coupled could not
detect even a fully matched target of 16 nt
(ODNfull(16)). However, in marked contrast,
the RNase H activity coupled system
allowed the 16-nt target to activate the
translation of the MB-riboregulator
mRNA (Ion/Ioff = 565 % for lane 5 versus
lane 4 in Figure 2 c) by concomitant cataFigure 2. RNase H activity coupled sensing of the human CCR5 sequence by CL assay for a
lytic cleavage of the hybridized MB–
2.5-mL aliquot of the RNase H activity coupled or noncoupled translation system (10 mL)
mRNA probe. Importantly, as expected,
after treatment with luciferase assay solution (100 mL; Promega). a) and b) Relative CL
the shorter 16-nt-targeting MB–mRNA
intensities in the presence or absence of target ODNs (1.8 pmol, target site 620–637) with
system, coupled with RNase H activity,
a) MB–mRNA (1.8 pmol) or b) dsDNA template (0.2 pmol) as a probe. c) Relative CL
intensities in the presence of target ODN(16) (1.8 pmol, target site 620–635) with MB–
succeeded in discriminating even the C to
mRNA probe (1.8 pmol). d) Relative CL intensities in the presence of target ODNs
T transition at position 627 (ODN1misT(16),
(45 fmol; target site: 620–637) with MB–mRNA probe (0.45 pmol).
lane 5 versus lane 6 in Figure 2 c).
The advantage of the present RNase H
activity coupled system is greatly emphasized when we detect a small amount of target DNA.[19, 20] The
sensitivity or detection limit related to the Ion/Ioff value was
enhanced by the RNase H activity coupled MB–mRNA
system, wherein the translation-activated mRNA probe
could be catalytically amplified above the equivalent stoichiometry of the target through the third catalytic process. While
the RNase H free system failed to detect 45 fmol (18 nm,
2.5 mL) of target ODNfull (lane 3 versus lane 4 in Figure 2 d),[21]
the RNase H activity coupled system enabled clear detection
of the same amount of target with a CL enhancement of about
400 % (Ion = 24.7 C 107 cpm (counts per minute), Ioff = 6.5 C
107 cpm) in combination with a sensitive luminometer
(Lumat LB 9507; lane 1 versus lane 2 in Figure 2 d). This
Figure 3. Detection of the nucleotide 21–36 region of the CCR5
triple catalytic system allows detection of 9 fmol (3.6 nm,
sequence with the designed MB–mRNA probe (1.8 pmol) by a CL
2.5 mL) of target with repetitively reproducible results in the
CL enhancement (Ion/Ioff = 1.7; data not shown).
Finally, we achieved visible sensing of the target sequence
by an unmodified but functional RNA probe. To visualize the
Two-base-mismatched ODN2mis (T for C and A for T at
sensing signal, we prepared a color-reporting MB–mRNA
positions 627 and 628) and one-base-mismatched ODN1misA
probe by exchanging the gene encoding luciferase with that
(C to A transversion at position 627) gave no notable
for b-galactosidase (b-Gal; target: nucleotide 620–637 region
enhancement (Ion/Ioff = 127 % for lane 3 versus lane 5 and
of the CCR5 gene sequence). The expressed b-Gal could
170 % for lane 3 versus lane 7, respectively, in Figure 2 a).
convert the substrate ortho-nitrophenyl-b-d-galactopyranoHowever, nonnegligible CL enhancement was observed for
side (ONPG) into ortho-nitrophenol (ONP), hence leading to
the one-base-mismatched target ODN1misT (C to T transition
a visible color change to light yellow. Transcription/transat position 627), perhaps due to the stable GT mismatch base
lation of the dsDNA template for the MB-controlled mRNA
pair in the resulting heteroduplex (Ion/Ioff = 424 % for lane 3
for b-Gal (0.2 pmol) was carried out in the presence of
versus lane 6 in Figure 2 a).
Angew. Chem. 2006, 118, 2945 –2949
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
RNase H (0.1 U mL 1) and target ODNfull (1.8 pmol). Enzymatic conversion of ONPG into ONP was quantitatively
evaluated by measuring the absorbance increase at 405 nm
(Figure 4 a). In a similar manner to that described above, the
method may facilitate high-throughput multiplex screening of
genetic sequences (by using fluorescent proteins with different emission properties) or be applicable in electrochemical
sensing devices (by using electrochemically sensible redox
proteins). Nucleic acid based nanomachines or nanodevices
may also be a potential application of this approach.[22]
Further work is now under way along these lines.
Received: October 31, 2005
Revised: January 16, 2006
Published online: March 21, 2006
Keywords: DNA recognition · molecular beacons · nucleic acids ·
RNA · sensors
Figure 4. Visible sensing of the CCR5 gene sequence (region 620–637)
by a b-galactosidase-encoding MB–mRNA probe. a) Absorbance
(405 nm) of b-galactosidase assay solution with ONPG as a substrate
(100 mL) containing an 11-mL aliquot of RNase H (0.1 U mL 1) activity
coupled MB–mRNA system (0.2 pmol of dsDNA template) translated
in the presence of target ODNfull (1.8 pmol; target site: nucleotide 620–637 region of the human CCR5 gene). b) Reaction scheme
and photographic images of the assay solutions.
remarkably high absorbance increase (Ioff = 0.04 for lane 3,
Ion = 0.59 for lane 4) was obtained, as expected, only for the
RNase H coupled translation system; Ion/Ioff reached 1475 %
(Figure 4 a, lane 4 versus lane 3). Detection could be readily
achieved with the naked eye. The presence of target ODNfull
was easily distinguished by the visible yellow color of the
solution (Figure 4 b, lane 3 versus lane 2).
In summary, we have demonstrated catalytic sensing of
nucleic acids by using an unmodified RNA probe in an
RNase H activity coupled cell-free translation system. The
combination with RNase H activity, which induces the third
signal-amplifying process of catalytic and irreversible activation of an mRNA probe, achieves an improved sequence
selectivity (1-nucleotide selectivity in a target ODN of 16 nts
in length) and an enhanced sensitivity ( 9 fmol of target).
The observed sensitivity is comparable with that of the
previously reported isothermal sensing system with an
RNase H activity coupled, cleavable, fluorescence resonance-energy transfer (FRET) probe,[15c] but should also be
compared with much higher sensitivities developed recently.[2f] Nevertheless, the characteristic aspect of the present
system that simple/unmodified RNAs or even dsDNAs can be
used as probes is something that can not be mimicked readily
by other methods.
In particular, the present RNase H activity coupled
system also allowed visible sensing of the target sequence
under isothermal conditions. It is remarkable that the target
sequence can be color-visualized without fluorophore/
quencher molecules or inorganic nanoparticles such as
quantum dots. What is needed for this color-based sensing is
simply a genetically encodable unmodified RNA that works
in a prokaryotic translation system. Since, in principle, any
type of reporter proteins can be generated, the present
[1] K. Nakatani, ChemBioChem 2004, 5, 1623, and references
[2] For examples of homogeneous gene sensing, see: a) S. Tyagi,
F. R. Kramer, Nat. Biotechnol. 1996, 14, 303; b) S. Tyagi, D. P.
Bratu, F. R. Kramer, Nat. Biotechnol. 1998, 16, 49; c) R. T.
Ranasinghe, L. J. Brown, T. Brown, Chem. Commun. 2001, 1480;
d) S. Sando, E. T. Kool, J. Am. Chem. Soc. 2002, 124, 2096; e) J.
Brunner, R. Kraemer, J. Am. Chem. Soc. 2004, 126, 13 626;
f) N. L. Rosi, C. A. Mirkin, Chem. Rev. 2005, 105, 1547; g) C. J.
Yang, H. Lin, W. Tan, J. Am. Chem. Soc. 2005, 127, 12 772.
[3] a) M. N. Stojanovic, P. de Prada, D. W. Landry, ChemBioChem
2001, 2, 411; b) M. N. Stojanovic, D. Stefanovic, Nat. Biotechnol.
2003, 21, 1069; c) Y. Tian, C. Mao, Talanta 2005, 67, 532.
[4] a) J. S. Hartig, I. GrHne, S. H. Najafi-Shoushtari, M. Famulok, J.
Am. Chem. Soc. 2004, 126, 722; b) S. H. Najafi-Shoushtari, G.
Mayer, M. Famulok, Nucleic Acids Res. 2004, 32, 3212.
[5] S. Sando, T. Sasaki, K. Kanatani, Y. Aoyama, J. Am. Chem. Soc.
2003, 125, 15 720.
[6] A. Saghatelian, K. M. Guckian, D. A. Thayer, M. R. Ghadiri, J.
Am. Chem. Soc. 2003, 125, 344.
[7] V. Pavlov, B. Shlyahovsky, I. Willner, J. Am. Chem. Soc. 2005,
127, 6522.
[8] H. Abe, E. T. Kool, J. Am. Chem. Soc. 2004, 126, 13 980.
[9] J. Cai, X. Li, X. Yue, J. S. Taylor, J. Am. Chem. Soc. 2004, 126,
16 324.
[10] C. Fan, K. W. Plaxco, A. J. Heeger, Proc. Natl. Acad. Sci. USA
2003, 100, 9134.
[11] F. Patolsky, Y. Weizmann, E. Katz, I. Willner, Angew. Chem.
2003, 115, 2474; Angew. Chem. Int. Ed. 2003, 42, 2372.
[12] S. Sando, A. Narita, K. Abe, Y. Aoyama, J. Am. Chem. Soc. 2005,
127, 5300.
[13] S. Brantl, Biochim. Biophys. Acta 2002, 1575, 15.
[14] F. J. Isaacs, D. J. Dwyer, C. Ding, D. D. Pervouchine, C. R.
Cantor, J. J. Collins, Nat. Biotechnol. 2004, 22, 841.
[15] For an example of a nucleic acid detection system that utilizes
RNase H, see: a) P. Duck, G. Alvarado-Urbina, B. Burdick, B.
Collier, BioTechniques 1990, 9, 142; b) L. Cloney, C. Marlowe,
A. Wong, R. Chow, R. Bryan, Mol. Cell. Probes 1999, 13, 191;
c) J. J. Harvey, S. P. Lee, E. K. Chan, J. H. Kim, E.-S. Hwang, C.Y. Cha, J. R. Knutson, M. K. Han, Anal. Biochem. 2004, 333, 246.
[16] For details on nucleotide numbering, see: S. Mummidi, S. S.
Ahuja, B. L. McDaniel, S. K. Ahuja, J. Biol. Chem. 1997, 272,
30 662.
[17] Y. Shimizu, A. Inoue, Y. Tomari, T. Suzuki, T. Yokogawa, K.
Nishikawa, T. Ueda, Nat. Biotechnol. 2001, 19, 751.
[18] We independently confirmed that MB–mRNA-based nucleic
acid sensing can also be carried out in a normal Eschericha coli
S30 extract system (RTS HY100, Roche; 10 mL): intensity of
luminescence with translation on/intensity of luminescence with
translation off (Ion/Ioff) = 400–600 % for the MB–mRNA probe
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. 2006, 118, 2945 –2949
(1.8 pmol) with fully matched target ODNfull (20 pmol). However, addition of Tth RNase H induced no signal enhancement in
this non-reconstituted cell-extract system.
We carried out RNase H induced cleavage reactions of an MB
domain with the 1–92 nucleotide sequence of the MB–mRNA
probe used here. Gel analysis (see Supporting Information)
indicated that cleavage occurred only after incubation in the
copresence of the RNase H and target ODN, a result indicating
that RNase H indeed catalyzed the target-assisted cleavage of
the RNA probe with a turnover number of 50 with respect to
the target ODN.
A [target-]dependence study (see Supporting Information)
revealed that the RNase H assisted MB–mRNA probe was
70 % fully activated even in the presence of 0.1 equiv of target
ODN (probe/target = 10), in contrast to the activity of a stemfree control probe which existed in an open or translation-on
state constantly in the absence of the target. This overstoichiometric activation of the probe may be most reasonably
explained in terms of target-assisted catalytic cleavage of the
probe by RNase H.
As described in our previous report,[12] the original MB–
luciferase mRNA probe (4.5 fmol) allowed detection of
50 fmol of target. However, the signal intensity obtained is
quite low compared with that of the present RNase H activity
coupled system, due to the absence of the third signal-amplifying
process of catalytic activation of the mRNA probe.
N. C. Seeman, Trends Biochem. Sci. 2005, 30, 119, and references
Angew. Chem. 2006, 118, 2945 –2949
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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