www.elsevier.com/locate/yabio SigniWcant enhancement of Xuorescence on hybridization of a molecular beacon to a target DNA in the presence of a site-speciWc DNA nickas

Damir S. Kopein A

Evgeniy A. Rogulin A

Ludmila A. Zheleznaya A

Nikolay I. Matvienko C

Sergey I. Gubanov B

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ANALYTICAL
BIOCHEMISTRY
Analytical Biochemistry 348 (2006) 123–126
www.elsevier.com/locate/yabio
0003-2697/$ - see front matter   2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2005.10.024
SigniWcant enhancement of Xuorescence on hybridization of a molecular 
beacon to a target DNA in the presence of a site-speciWc DNA nickase
Ludmila A. Zheleznaya a, Damir S. Kopein a, Evgeniy A. Rogulin a, 
Sergey I. Gubanov b, Nikolay I. Matvienko c,¤
a Institute of Theoretical and Experimental Biophysics, 142290 Poushchino, Russian Federation
b Bioline GmbH, 14943 Luckenwalde, Germany
c Institute of Protein Research, 142290 Poushchino, Russian Federation
Received 12 August 2005
Available online 2 November 2005
Abstract
We have developed a simple isothermal (55°C) reaction that permits detection of DNA targets using only two components: a molecu-
lar beacon and a site-speciWc DNA nickase without deoxyribonucleotide triphosphates and primers. The loop sequence of the molecular
beacon should contain a DNA nickase recognition site. The nickase–molecular beacon (NMB) combination permits a 100-fold increase
in Xuorescent signal. The applications of the NMB assay for enhancement of Xuorescent signal in some isothermal methods are discussed.
  2005 Elsevier Inc. All rights reserved.
Keywords: Molecular beacon; Site-speciWc DNA nickase; Rolling-circle ampliWcation
As Wrst described, molecular beacons are synthetic DNA
and RNA probes that Xuoresce on hybridization [1]. A
molecular beacon is a single-stranded oligonucleotide that
has a Xuorescent moiety at one end and a nonXuorescent
quencher at the other end. The oligonucleotide contains a
sequence complementary to a target that is Xanked by short
complementary sequences. In the absence of a speciWc sin-
gle-stranded target sequence, the complementary sequences
of the molecular beacon anneal to form a stem–loop struc-
ture that brings the two moieties into close proximity to
each other, causing the Xuorescence of the Xuorophore to
be quenched by Xuorescence resonance energy transfer
(FRET).1 Hybridization of the loop to a target sequence
leads to a separation of the Xuorophore and the quencher
from each other and Xuorescence is not quenched.
Molecular beacons are increasingly used in many appli-
cations, including quantitative PCR, single nucleotide poly-
morphism (SNP) analysis, visualization of RNA expression
in living cells, and detection of DNA binding proteins [2].
In all methods to detect speciWc nucleic acid sequences, a
Xuorescent signal is proportional to the amount of target
sequence that is ampliWed during the reaction. In this arti-
cle, we describe a new method whereby the amount of tar-
get remains constant throughout the reaction. In this case,
the increase in Xuorescent signal is due to cleavage of the
molecular beacon loop by a site-speciWc DNA nickase.
Site-speciWc DNA nickases, like type II restriction
endonucleases, recognize short-speciWc sequences on dou-
ble-stranded DNA. However, in contrast to restriction
endonucleases, nickases cleave only one strand. Today, four
nickases have been isolated from natural sources [3], and
DNA-nicking derivatives of some restriction endonucleases
have been obtained by random mutagenesis [4], by selection
from randomized expression library [5], and by catalytic
* Corresponding author.
E-mail address: nikmatv@vega.protres.ru (N.I. Matvienko).
1 Abbreviations used: FRET, Xuorescence resonance energy transfer;
SNP, single nucleotide polymorphism; PNA, peptide nucleic acid; DTT,
dithiothreitol; BSA, bovine serum albumin; NMB, nickase–molecular bea-
con; RCA, rolling-circle ampliWcation; HRCA, hyperbranched rolling-
circle ampliWcation.124 Enhancement of Xuorescence in the presence of DNA nickase / L.A. Zheleznaya et al. / Anal. Biochem. 348 (2006) 123–126
site mutagenesis of the subunits of the heterodimeric
restriction endonuclease BbvCI [6]. An artiWcial DNA-
nicking system based on restriction enzymes assisted by
peptide nucleic acid (PNA) openers has also been devel-
oped  [7]. In the current work, we use DNA nickase
Nb.BspD6I that recognizes the double-stranded 5-
GAGTC-3/5-GACTC-3 site and cuts the GAGTC
strand at the fourth nucleotide downstream from the site
[8]. The nickase reveals an optimal activity at 55°C.
Here we demonstrate that up to 100 molecular beacons
can be cleaved by nickase on a single target molecule.
Materials and methods
Oligonucleotides
All oligodeoxyribonucleotides were obtained commer-
cially from Eurogentec. The sequences of the oligonucleo-
tides were as follows:
St5: 5-FAM-GGCATTCTAGAGTCGACCTGCAGG
CATGATGCC-BHQ1-3
St7: 5-FAM-GGCATCTTCTAGAGTCGACCTGCA
GGCATGAGATGCC-BHQ1-3
St9: 5-FAM-GGCATCTATTCTAGAGTCGACCTG
CAGGCATGATAGATGCC-BHQ1-3
C5: 5-GGCATCATGCCTGCAGGTCGACTCTAG
AATGCC-3
C7: 5-GGCATCTCATGCCTGCAGGTCGACTCTA
GAAGATGCC-3
C9: 5-GGCATCTATCATGCCTGCAGGTCGACTC
TAGAATAGATGCC-3
The oligonucleotides St5, St7, and St9 were molecular
beacons with stem lengths of 5, 7, and 9bp, respectively
(stem arms are underlined in the text). All of the molecular
beacons had the same loop sequence that was complemen-
tary to the polylinker region (6241–6263) of the phage
M13mp19 DNA. The region contains the 5-GACTC-3
sequence. 6-CarboxyXuorescein was labeled at the 5 end as
the Xuorophore, and a black hole quencher was labeled at
the 3 end as the quencher. The oligonucleotides C5, C7,
and C9 were perfectly complementary to the oligonucleo-
tides St5, St7, and St9, respectively.
DNA nickase
The N.BspD6I recombinant nickase was isolated from
the TOP10F (pRARE/Nick) Escherichia coli strain con-
taining two plasmids [9]. The plasmid pET28b/Nbsp car-
ried the nbsp gene under the phage T7 promoter. The
second plasmid, pRARE/Mssc, contained the DNA meth-
yltransferase M.SscL1I gene inserted into plasmid
pRARE. The M.SscL1I has been shown to protect bacte-
rial DNA against hydrolysis by the nickase [8]. pRARE
(Novagen) is a plasmid that contains genes of tRNA that
rarely occur in E. coli. PuriWed nickase (>95%) was
obtained using a phosphocellulose P11 (Whatman) col-
umn followed by hydroxyapatite HTP (Bio-Rad) and Ni-
NTA agarose (Qiagen) columns and had activity of
approximately 0.5£106U/mg. The enzyme is commer-
cially available from Bioline (Cat. No. BIO-27039, Lon-
don, UK).
DNAs
The single-stranded phage M13mp19 DNA was isolated
according to a standard procedure [10]. After quantitation
with a UV spectrophotometer, the DNA was serially
diluted in steps to a Wnal concentration of 1–0.01M. Ali-
quots of 1l were used in the reactions. Thymus DNA was
purchased from Sigma.
Nickase–molecular beacon assay
A premix for a number of reactions was prepared. Each
reaction (20l) contained (Wnal concentration) 1£ nickase
buVer (10mM Tris–HCl [pH 7.8], 150mM KCl, 10mM
MgCl2, 1mM dithiothreitol [DTT], 0.1g/l bovine serum
albumin [BSA]), 1l nickase (0.3mg/ml), 1l ssM13mp19
DNA, and a molecular beacon at diVerent concentrations
(0.5–5M). The reactions were followed in a Rotor–Gene
RG-3000 (Corbet Research) equipped with Rotor–Gene
6.0 for Windows.
Results and discussion
Previously, we showed that the nickase N.BstD6I can
cleave linear nonXuorescent oligonucleotides repeatedly on
a single target molecule provided that the target–oligonu-
cleotide hybrid has a nickase recognition site [8]. In the cur-
rent study, we used the same nickase in combination with
molecular beacons, allowing registration of the cleavage
process in real time.
Fig. 1 gives the concept view of the method. First, the
loop of the molecular beacon is complementary to a tar-
get DNA and contains the 5-GAGTC-3 sequence.
Consequently, the target should contain the 5-GACTC-
3 sequence. This limitation is not severe because DNA
sites featuring such sequence should occur quite
frequently; statistically, one should occur every 200–
300bp of a random DNA sequence, on the average.
Second, on hybridization of the loop to the target, the
double-stranded nickase recognition site is formed and a
nickase cuts the molecular beacon, producing two single-
stranded fragments. Third, the two fragments are
separated from the target if their melting temperatures are
lower than the reaction temperature (55°C). Conse-
quently, the cleavage must give rise to an irreversible
Xuorescence enhancement. Fourth, the target released
from the fragments is capable of adopting the next molec-
ular beacon; thus, the cleavage process will be repeated
many times on one target, providing accumulation of the
Xuorescence signal.Enhancement of Xuorescence in the presence of DNA nickase / L.A. Zheleznaya et al. / Anal. Biochem. 348 (2006) 123–126 125
To test this principle, we used three molecular beacons
with a loop length of 23 bases and stem lengths of 5, 7, and 9
bases. The length of the loop was chosen to ensure that the
molecular beacon–target hybrid would be formed at the
reaction temperature and that the fragments of the loop aris-
ing after cleavage by nickase would separate from the target.
Thermal denaturation proWle studies were conducted to
determine which of the molecular beacons is preferable for
using in nickase–molecular beacon (NMB) assays (Fig. 2).
The thermal transition proWles were in good accordance
with the data reported by Tsourkas and coworkers [11].
The molecular beacon with the 5-base stem was unstable at
55°C and had a great background. The molecular beacon
with the 9-nucleotide stem was not completely melted even
at 90°C and produced the lowest signal on hybridization
with complementary oligonucleotide. Therefore, for further
study we chose the molecular beacon with the 7-base stem.
Fig. 3A shows the time curves of Xuorescence intensity of
St7 during cleavage by nickase. It can be seen that in the
presence of a 50-nM target DNA, the Xuorescence rose rap-
idly at Wrst and then asymptotically approached a plateau.
The  Xuorescence enhancement reXects restoration of the
Xuorescence of the Xuorophore resulting from cleavage of
the molecular beacon. All molecular beacons were cleaved
after 25min of reaction. However, the Xuorescence intensity
falls rapidly with target concentration, and its time depen-
dence becomes linear. In the presence of 5nM target, the
intensity approaches a plateau in 16h of incubation with
nickase (data not shown). The sensitivity limit of the NMB
assay is 0.5nM. We found that changes in KCl concentra-
tions (50–300mM), in MgCl concentrations (5–20mM), and
in pH values (7.0–9.5) did not result in any increase in sensi-
tivity of the NMB assay. The limiting step seems to be slow
dissociation of nickase from the site. The Xuorescence inten-
sity increases with molecular beacon concentration. How-
ever, we could not use molecular beacon concentrations
greater than 5M due to the upper measuring limit of the
device. The addition of excess total calf thymus DNA did
not signiWcantly decrease the response (Fig. 3B). Therefore,
the NMB assay can be used to enhance the Xuorescence
intensity of a target being in a complex mixture of DNAs.
We deWne the Xuorescence enhancement factor as ratio of
the  Xuorescence intensity (background subtracted) of the
NMB assay after reaction to that (background subtracted) of
the hybrid (target–molecular beacon). The background is
Xuorescence intensity of the molecular beacon alone. The
enhancement factors were determined at diVerent ratios of the
molecular beacons to the constant amount of target (50nM).
These data are shown in Table 1. It can be seen that in the
presence of a 100-fold excess of the molecular beacon, the
enhancement factor of Xuorescence reaches 100 after 60min
incubation with the nickase. Therefore, at least 100 molecular
beacons can be cleaved on one target molecule during 60min.
Our NMB assay can be used to increase the Xuorescence
in isothermal ampliWcation of DNA at temperatures not
Fig. 1. Scheme demonstrating the use of a molecular beacon in hybridiza-
tion analysis of nucleic acids with site-speciWc nickases. (A) Hybridization
of the molecular beacon. (B) Cleavage of the molecular beacon by nickase.
(C) Separation of short fragments and nickase. (a) Xuorescence quencher;
(b) Xuorophore whose emission is absorbed by the quencher; (c) emitting
Xuorophore.
Fig. 2. (A) Melting curves of molecular beacons (0.5 M in 1£ nickase buVer) without any oligonucleotides or in the presence of 10-fold excess of oligonu-
cleotides (+) perfectly complementary to each molecular beacon. The temperature proWles were obtained by sampling Xuorescence after temperature
increments of 1°C held for 30s. (B) Enlarged version of marked area in panel A.126 Enhancement of Xuorescence in the presence of DNA nickase / L.A. Zheleznaya et al. / Anal. Biochem. 348 (2006) 123–126
exceeding 60°C if the target molecule includes the GACTC
sequence. In particular, it can be useful in real-time monitor-
ing of the rolling-circle ampliWcation (RCA) and the hyper-
branched rolling-circle ampliWcation (HRCA) [12,13]. The
nucleotide sequence of a molecular beacon loop that includes
a nickase site could be inserted in a padlock probe [14] out-
side the region complementary to the target. In this case, the
same molecular beacon can be used for the detection of
diVerent targets. The combination of the nickase and beacon
could also be used in the loop-mediated isothermal ampliW-
cation of DNA [15]. In this case, the molecular beacon loop
must be complementary to the ampliWed DNA loops having
arbitrary sequences. Unfortunately, the nickase N.BspD6I
does not cleave DNA–RNA hybrids [8]. Therefore, the NMB
assay cannot be used for detection of RNA targets.
Acknowledgments
This work was supported by the Russian Academy of
Sciences (Grant p2004naukograd 04-04-97313) and the
Russian Foundation of Base Research (projects 03-04-
48967, 04-04-08169, and 05-04-48901). The authors thank
Maxim V. Patrushev for helpful discussion.
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Table 1
Enhancement factors of NBM assays
Concentration 
of beacon St7
Time of reaction (min)
5 1 02 03 04 06 0 1 2 0
0.5M 4.71 13.18 16.11 16.76 17.20 17.39 17.47
1M 20.09 28.61 38.38 42.94 44.74 46.27 47.48
2.5M 22.69 33.90 52.70 64.43 76.21 89.15 109.08
5M 20.34 31.48 51.80 67.48 81.39 104.18 106.80
Fig. 3. Time course of cleavage of the molecular beacon St7 (0.5M) with the nickase. (A) The concentrations of target (ssDNA of M13mp19 phage) are
denoted by numbers above the curves. The arrow marks the time of the addition of nickase. (B) EVect of calf thymus DNA on the NMB assay. (a,b) 50nM
of target; (c,d) 5nM of target; (e,f) 0.5nM of target; (a,c,e) without 200g/ml calf thymus DNA; (b,d,f) with 200g/ml calf thymus DNA.

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