Long-lived luminogenic probe for detection of RNA in a crude solution of living bacterial cells

Article abstract of DOI:10.1021/ja406724k

A pre-type sensitizer for a lanthanide complex on an oligonucleotide was successfully converted to a perfect final structure in a target DNA/RNA-templated reaction, without any chemical reagent or enzyme, under neutral conditions. The final form of the lanthanide-oligonucleotide provided a long-lived luminescence signal, appropriate for time-gated luminescence analysis and signal amplification. Target DNA/RNA-assisted time-gated luminescence analysis is a powerful tool for elimination of autofluorescence and detection of target RNA in living bacterial cells.

Full text of DOI:10.1021/ja406724k

Communication
pubs.acs.org/JACS
Long-Lived Luminogenic Probe for Detection of RNA in a Crude
Solution of Living Bacterial Cells
Hisao Saneyoshi,‡,† Yoshihiro Ito,*,‡,⊥ and Hiroshi Abe*,‡,§,⊥
‡Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
§PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
⊥Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama
351-0198, Japan
S
* Supporting Information
chelator with lanthanide ion to obtain luminescence of a similar
level as the molecular beacon-type probes recently reported.7
ABSTRACT: A pre-type sensitizer for a lanthanide
complex on an oligonucleotide was successfully converted
to a perfect final structure in a target DNA/RNA-
templated reaction, without any chemical reagent or
enzyme, under neutral conditions. The final form of the
lanthanide−oligonucleotide provided a long-lived lumines-
cence signal, appropriate for time-gated luminescence
analysis and signal amplification. Target DNA/RNA-
assisted time-gated luminescence analysis is a powerful
tool for elimination of autofluorescence and detection of
target RNA in living bacterial cells.
However, lanthanide-based detection methods have been
applied only to the detection of RNA or DNA in vitro, and
have not been applied to the detection of cellular RNA.
Nucleic acids modified with gold nanoparticles have been
used to detect nucleic acids. Sensitive detection at a femtomolar
level was achieved in vitro through the measurement of light
scatter.8 Fluorogenic gold nanoparticles have been used to
detect RNA in living cells.9 The great benefit of gold
nanoparticles is their cell membrane permeability.
Conversely, lanthanide probes with luminogenic properties
and long-lived luminescence could provide high S/B ratios, by
removing autofluorescence, for the detection of cellular RNA,
although these probes require transfection reagents for
introduction into cells. To solve the problems associated with
previous lanthanide-based oligonucleotide probes, including the
inability to amplify signals, we designed a new chemical-
reaction-triggered luminogenic DNA probe. A DNA-templated
chemical reaction could offer significant advantages, such as
high S/B ratio and signal amplification for detection. Various
DNA-templated fluorogenic chemical reactions have been
reported.10 However, no chemical-reaction-triggered, lantha-
nide-based luminogenic probe has been reported. A key issue is
how to make a switch for lanthanide-based luminogenic
systems. As the switching system, Terai et al.4e proposed a
photoinduced electron transfer (PeT) system, using aniline
attached onto 7-amino-4-methyl-2(1H)-quinolinone (carbos-
tyril 124), which was previously used for a labeling study.11
They demonstrated that the PeT mechanism could be used as
an ON-OFF switch, and they successfully applied it to a long-
lived protease probe.4d For highly sensitive probes for DNA/
RNA sensing applications, an ON switch is needed. One
possibility, proposed by the same authors, is to use an antenna
molecule. The key to this mechanism is the structural change of
the antenna molecule,12a initiated by Skraup reaction between
acrolein and the aniline derivative, to form a quinoline skeleton
as an antenna molecule. This absorbs a much longer
wavelength than the starting aniline derivative. The antenna
molecule is specifically excited to transfer energy to the
lanthanide complex. Quite recently, Pershagen et al.12b reported
etection and imaging of DNA/RNA species in native cells
D
are gaining importance in biological and biomedical
research. However, contamination by autofluorescence disturbs
the desired signal. Thus, elimination of severe autofluorescence
from biological samples is important for performing sensitive
and simple experiments. Generally, the biological environment
has several autofluorescent components, such as NADH, FAD,
and riboflavin. To reduce the effect of autofluorescence, and
thus obtain high signal/background (S/B) ratios, long-lived
luminescent compounds may be useful. These can be easily
discriminated in terms of large differences in fluorescence
lifetime.1,2
Time-gated fluorescence measurement using organic fluo-
rescent compounds has been applied to in vitro detection of
RNA to overcome autofluorescence.3 However, organic
fluorescent compounds provide only a slightly longer
fluorescence lifetime than autofluorescence. For example, a
pyrene excimer had a 40 ns lifetime, compared with 7 ns of
autofluorescence from cellular extracts. In contrast, lanthanide
complexes offered a much longer luminescence lifetime (in the
order of milliseconds). Lanthanide complexes have been
successfully employed in sensing metal ions, monitoring
enzymatic activity, and detecting proteins in living cells, time-
gated spectroscopy, microscopy, and flow cytometry.4
In the field of nucleic acid detection, Oser et al. proposed the
use of lanthanide-based probes and applied them to template-
mediated formation of lanthanide complexes for the first time.5
Later, several chemists reported template-mediated lanthanide
formation for sensing target nucleic acid sequences.6 These
split-type probes depend on the hybridization affinity and an
appropriate distance between the light-harvesting group and
Received: July 2, 2013
© XXXX American Chemical Society
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that an acyclic coumarin precursor was converted into an active
antenna, triggered by analytes such as Pd0/2+, H2O2, F−, and
enzymes. However, to apply this system to our oligonucleotide
probe, we ideally needed neutral reaction conditions and no
requirement for additional chemicals or enzymes.
Here, we synthesize a new antenna molecule using the
Staudinger reaction and apply it to development of a
lanthanide-based luminogenic DNA probe. This probe enables
sensitive detection of DNA or RNA by time-gated
luminescence analysis, as shown in Figure 1. We demonstrate
direct detection of RNA in living Escherichia coli cells by
measuring samples in a cuvette.
new antenna structure was confirmed by UV absorbance
following cyclization under reductive conditions (Figure S2).
Figure S2 shows that phenyl azide group 4 was reduced by tris-
carboxyethylphosphine (TCEP) to a cyclized species, phenan-
thridinone derivative 4′. The cyclization reaction ceased after
200 s, as shown in Figure S2.
For the synthesis of the target probe, aniline derivative 1 was
chosen as a starting material. The amino group was converted
into triazene for protection and pre-activation.15 The triazene
derivative 2 was assembled with o-methoxybenzoylboronic acid
using Suzuki coupling conditions, to give biaryl compound 3.
Subsequently, the triazene group was rapidly converted into
azide derivative 4 by reaction with trimethylsilyl azide
(TMSN3) under acidic conditions.
In Scheme 1, methyl benzoate derivative 4 was treated with
lithium hydroxide, and then the carboxyl group was
Scheme 1. Synthesis of Pre-antenna-Chelator Complex
Figure 1. (A) Chemical-reaction-triggered luminogenic reaction. (B)
Detection of RNA species in the crude solution of bacterial cells by
time-gated luminescence analysis.
For design of the antenna molecule to be switched by
chemical reaction, we separated 2-quinolinone, which is the
core structure of carbostyril 124, as shown in Figure S1. When
the lactam structure was divided at the amide moiety into ester
and amine, no emission was expected. Previous studies have
reported the generation of fluorescence by coumarin or
quinolinone, starting from o-hydroxy- or o-amino-cinnamate
derivatives via double bond isomerization by photoirradiation.13
The Z-isomer is the critical conformation for the formation of
the lactam structure to recover the active antenna molecule
structure. However, it is thermally unstable and light sensitive,
and rapidly isomerizes to the E-isomer, which cannot be
converted to a lactam ring under neutral conditions at ambient
temperature because the reactive groups are too distant.
To solve this problem, we conformationally locked the active
geometry to add a benzene ring to the double bond moiety for
cyclization between ester and amine, as shown in Figure S1.
The o-amine(de)−o-ester pair on the biphenyl skeleton is
highly reactive and forms a phenanthridinone ring.14 To control
autocyclization, the amino group was caged as an azide group.
After reduction of the azide group with a reducing reagent such
as phosphine, we expected to obtain an active antenna structure
to excite the lanthanide chelate. The expanded-ring version of
2-quinolinone, phenanthridinone, should have a similar UV
absorption wavelength and act as the antenna molecule,
stimulating the lanthanide complex. The formation of the
immediately coupled with mono-o-nitrobenzenesulfonyl (Ns)-
protected ethylenediamine hydrochloride, using 4-(4,6-dime-
thoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as a
condensation reagent to give 5. A linker moiety for conjugation
with the oligonucleotide probe was attached at the sulfone
amide by alkylation, followed by the deprotection of the Ns
group with thiophenol.16 Diethylenetriaminepentaacetic acid
(DTPA) was selected as a chelator because of its high stability
for lanthanide ions.17 Diethylenetriaminepentaacetic anhydride
was coupled with secondary amine 7 to give compound 8.
A model luminogenic compound (8′) was synthesized from
8 by treatment with EuCl3 in MeOH in the presence of NaOH.
UV spectra and time-gated luminescence spectra clearly showed
the conversion to a lactam structure (8″) and a 1500-fold
increase in luminescence, relative to the acyclic form, upon
reduction, as shown in Figure 2.
For the synthesis of luminogenic DNA-Az probe (Ln-Az),
the Boc group of compound 8 at the alkyl linker moiety was
removed under acidic conditions and then coupled with
bromoacetyl NHS ester to give the reagent for conjugation.
Oligonucleotide-3′-phosphorothioate was synthesized by DNA
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Figure 4. Time course of luminogenic reaction with full-match DNA,
mismatch DNA, and without template. Conditions: 50 mM MOPS
(pH 7.0), 100 mM NaCl, 10 μg/mL BSA, λEx 340 nm, delay time 0.05
ms, gate time 3 ms, slit width, 10 nm. Reaction was monitored at λEm
615 nm. Ln-Az probe (Eu3+): 5′-GCCGGCGG-Ln-Az (Eu3+)-3′, 250
nM. TPPc probe: 5′-TPPc-TGTGGGCA-3′, 500 nM. Match-DNA:
3′-CGCGGCCGCCACACCCGTTC-5′, 250 nM. Mismatch-DNA:
3′-CGCGGCCGCCACAGCCGTTC-5′, 250 nM.
Figure 2. Spectra of luminogenic compound 8′ and luminescent
compound 8″. Left: UV spectra. Right: Time-gated luminescence
spectra. UV spectrum conditions: 50 μM probe 8′, 50 mM MOPS
(pH 7.0), 20 mM TCEP. Time-gated luminescence spectra conditions:
1 μM probe, 50 mM MOPS, 2 mM TCEP, λEx 340 nm.
probes were designed to target 23S rRNA. Probes (Match pair
or Scramble pair) and cells were mixed in buffer in a cuvette
and directly analyzed by fluorescence spectrometry. The
autofluorescence from E. coli and the luminescent signal
overlapped in the 400−600 nm range and could not be
distinguished without time-gated monitoring, as shown in
Figure 5A. However, using a wait time of 0.1 ms,
synthesizer and coupled with bromoacetylated coupling unit 9.
In addition, Ln-Az probe was treated with TCEP to give the
reduced Ln-Az probe (rLn-Az). Each probe was mixed with
lanthanide ion (Eu3+ and Tb3+) for use in photophysical
studies, as shown in Figures 3 and S3 and Table S1.
Figure 5. Detection of 23S rRNA in E. coli cells by direct measurement
of cuvette sample: (A) no delay time and (B) 0.1 ms delay time.
Conditions: (E. coli, JM109) 1OD, 50 mM MOPS (pH 7.0), 1 M
NaCl, 0.05% SDS, λEx 340 nm, delay time 0−0.1 ms, gate time 6 ms,
slit width 10 nm. Ln-Az probe (Tb3+): 5′-CTGGCGGTCTGGGTT-
Ln-Az (Tb3+)-3′, 100 nM. TPPc probe with matched sequence: 5′-
TPPc-GTTTCCCTCTTCACG-3′, 200 nM. TPPc with scramble
sequence: 5′-TPPc-GCCTTCTCCCGAAGT-3′, 200 nM.
Figure 3. Structure of Ln-Az (open form) and rLn-Az probe (closed
form) and their spectral properties.
The spectra of the new Ln-Az probe (open form) show that
the open form did not emit fluorescence at 340 nm excitation.
However, the rLn-Az probe (closed form) effectively emitted
luminescence in the 490−700 nm range, depending on the
central metals in the chelator (Figure 3). Maximum emission of
the Eu3+ and Tb3+ probes was observed at 615 and 545 nm,
respectively. The lifetimes of luminescence of these probes
were 0.6 and 1.8 ms, respectively, which are sufficiently long to
eliminate background fluorescence, as shown in Figure S5 and
Table S1.18 The lanthanide complex was relatively photostable
under consecutive irradiation, compared with an organic
fluorescent dye, pyrene (Figure S4).
Next, to know whether Ln-Az probe can discriminate a single
base difference, detection of the ras gene was performed in vitro
using time-gated spectroscopy. In the presence of target
sequence, activation of Ln-Az (Eu3+) probe by reduction of
the triphenylphosphine carboxamide (TPPc)−oligonucleotide
probe immediately generated a long-lived luminescence signal
(Figure 4). On the other hand, experiments with mismatch-
containing target or without target showed no luminescence.
The maximum S/B ratio after 10 min was 400. The most
intriguing property, compared with previous lanthanide-based
oligonucleotide probes, was the ability to amplify signal during
the detection process (Figure S6). We confirmed that the probe
could amplify signal by at least 10-fold when a 100-fold excess
of probe versus target DNA was used, as described in Figure S7.
Finally, the direct detection of RNA species in crude solution
of living E. coli cells was performed. Ln-Az (Tb3+) and TPPc
autofluorescence was reduced to negligible levels (Figure 5B),
and the desired signal was observed with a high S/B ratio.
Further, we tested detection of DNA sequences in solutions
containing a high concentration of bovine serum albumin
(BSA) or 10% fetal bovine serum (Figure S8A−D). In both
solutions, no signal was detected in no-delay mode, but a
distinct signal was observed in time-gated mode. Ln-Az probe
was stable in these solutions since no significant increase of
signal was observed without target DNA during the time course
measurement (Figure S8E,F). Thus, we confirmed that time-
gated luminescence analysis using these probes could
sensitively detect RNA signals in biological samples. Although
other fluorescence-based methods can detect RNA in bacterial
cells by fluorescence microscopy,10l,p the direct detection of
RNA in a crude solution of bacterial cells by fluorescence
spectrometry has never been reported.
In this paper, we first designed a new phenanthridinone
derivative (8″ in Figure 2 or 4′ in Figure S2) that functioned as
an antenna molecule. This means that the excited phenan-
thridinone prefers to transition from a singlet state to a triplet
state. Based on this result, we designed a chemically switched
antenna molecule (9) that was derivatized from phenanthridi-
C
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Experimental details and NMR spectra of all new compounds,
Figures S1−S8, Scheme S1, and Table S1. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
y-ito@riken.jp
■
h-abe@pharm.hokudai.ac.jp
Present Address
†H.S.: Department of Material and Life Chemistry, Faculty of
Engineering, Kanagawa University, 3-27-1 Rokkakubashi,
Kanagawa-ku, Yokohama 221-8686, Japan
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
H.A. was financially supported by the Ministry of Education,
Culture, Sports, Science, and Technology (MEXT), Precursory
Research for Embryonic Science and Technology (PREST),
and the New Energy and Industrial Technology Development
Organization (NEDO). H.S. financially supported by a Grant-
in-Aid for Young Scientists (B). We are grateful for the support
received from the Brain Science Institute (BSI) Research
Resource Center for mass spectrum analysis.
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