Sandwich probes: Two simultaneous reactions for templated nucleic acid detection

Article abstract of DOI:10.1039/c0cc01968b

Fluorescence-quenched nucleic acid probes with reactive moieties at both the 5′ and 3′ ends are synthesized and tested for reaction with two adjacent nucleophile-containing DNAs. These probes improve signal to background over singly reactive probes and ca

Full text of DOI:10.1039/c0cc01968b

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Sandwich probes: two simultaneous reactions for templated nucleic acid
detectionw
Daniel J. Kleinbaum and Eric T. Kool*
Received 19th June 2010, Accepted 14th September 2010
DOI: 10.1039/c0cc01968b
Fluorescence-quenched nucleic acid probes with reactive
0
0
moieties at both the 5 and 3 ends are synthesized and tested
for reaction with two adjacent nucleophile-containing DNAs.
These probes improve signal to background over singly reactive
probes and can discriminate single nucleotide polymorphisms in
the target DNA or RNA.
Templated reactions have been studied increasingly as a
1
strategy for detection of nucleic acids in vitro and directly
–5
6
–10
in cells.
One of these approaches, quenched autoligation
(
QUAL) reactions, relies on an S 2 reaction between an
N
0
11,12
oligonucleotide with a 3 -nucleophile and a quenched probe
0
with a 5 -leaving group.
The reaction releases the quencher
and unquenches a nearby fluorophore, producing a signal. As
with virtually all DNA detection methods, background signals
can interfere at low analyte concentrations; in the case of
QUAL probes, background signals can arise from less-than-
complete initial quenching and from nonspecific hydrolysis.
Herein we describe a new reaction design for quenched
autoligation probes and for DNA-templated reactions in
general that can lower such sources of background. All
previous approaches to templated nucleic acid detection have
involved two probes binding adjacent to one another, bringing
functional groups into proximity for reaction. Here we report
a new geometry for templated reactions. In this new approach,
a labeled oligonucleotide is functionalized with electro-
philically-linked quenchers at both ends. Two activating
nucleophile probes bind on either side of this quenched probe,
performing simultaneous reactions to elicit a signal. We find
that this double-reaction approach produces a lower initial
background signal as well as a higher signal-to-background
than earlier single-reaction strategies, and is capable of detecting
rRNA in intact bacterial cells.
Fig. 1 Schematics and structures of Sandwich probes. (A) Sandwich
probes require two quencher displacement reactions (one at each end
of the probe) to fully unquench the fluorophore and produce a signal.
(B) Sequences of template, nucleophile and Sandwich probe oligo-
nucleotides for in vitro experiments. The template corresponds to a
sequence in bacterial rRNA and the base in bold is a position known
to vary among different species. (C) Structure of Sandwich probe and
0
0
expected reactions with adjacent 5 and 3 nucleophile-modified
oligonucleotides.
1
2
0
previously described butyl linker. At the 3 -end, a new linker
was needed to present an electrophilically-linked quencher and
allow for subsequent solid-phase DNA synthesis following the
coupling of the linker to the solid support. To fulfill these
criteria, a five-carbon linker was synthesized that contained
In this new templated reaction design, the doubly-
a dabsylate quencher, DMT-protected alcohol, and a
functionalized quencher oligonucleotides are flanked by both
0
phosphoramidite group. After coupling this linker to the solid
support through standard phosphoramidite chemistry, the DMT
0
a 5 -nucleophile oligonucleotide and a 3 -nucleophile oligo-
nucleotide in order for the fluorophore to be fully unquenched
0
0
group could be removed and standard 3 to 5 solid-phase DNA
synthesis could then proceed. The dabsylate group was stable to
DNA synthesis conditions, as evidenced by the persistent bright
orange color of the beads (and later characterization). The
(
Fig. 1); thus they are referred to as ‘‘Sandwich’’ probes. For
our experiments, the central quenched probe is a fluorescein-
labeled 10-mer designed to be highly responsive to single
nucleotide mismatches. The quencher-leaving groups at both
0
synthesis of this 3 -linker and the Sandwich oligonucleotide are
0
1
1
0
ends of the quenched probe are ‘‘dabsylate’’ moieties. The
0
described in the ESI,w as are the syntheses of the 3 and 5
dabsylate at the 5 -end of the probe was conjugated using a
nucleophile oligonucleotides. The structure of the Sandwich
probe is shown in Fig. 1C; the 2-cyanoethyl group was purposely
0
not cleaved from the 3 -phosphate group of the probe to reduce
Department of Chemistry, Stanford University, Stanford, CA 94305,
USA. E-mail: kool@stanford.edu; Fax: +1 650-725-0259;
Tel: +1 650-724-4741
w Electronic supplementary information (ESI) available: Experimental
details, additional figures, characterization of synthetic intermediates.
See DOI: 10.1039/c0cc01968b
the probability of an intramolecular reaction between the
13
phosphate and the quencher leaving group. The presence of
the three conjugated groups in the quenched probe, as well as
the intact cyanoethyl group, was confirmed by MALDI-mass
spectrometry (see ESIw).
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154 Chem. Commun., 2010, 46, 8154–8156
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Following the synthesis of the Sandwich probe, its initial
background fluorescence was measured and compared to
0
0
control probes containing either a single 5 or 3 dabsylate
modification alone (Fig. S1A, ESIw). The results showed that
the Sandwich probe exhibits better quenching in the unreacted
state than either single dabsyl probes by between two- and
six-fold.
Having a prototype Sandwich probe in hand, we proceeded
to test whether it could undergo DNA-templated reactions in
the presence of nucleophile-substituted probes. The displacement
reactions were monitored by following the increase in
fluorescence intensity of the reactions over time. The reactions
0
0
were performed with both 5 and 3 nucleophile DNAs, and
0
0
with controls containing either the 5 or 3 nucleophile DNAs
separately. We also tested the effect of the template DNA
explicitly by carrying out reactions with the three reacting
probes but in the absence of template (Fig. 2A).
The experiments showed that the presence of both nucleo-
philes in the templated reaction produced a more rapid
turn-on signal than with either individual nucleophile alone,
which establishes that both nucleophiles play a cooperative
role in developing the turn-on signal. The separate reactions
0
0
0
with the 5 or 3 nucleophiles revealed that the 3 -nucleophile
0
(
reacting with 5 dabsylate) is the faster reaction (by a factor of
B2–3 after four hours; Fig. 2A), which may be due to
differences in geometry and electrophilic linker structures.
Significantly, in the absence of template, reaction of the three
probes was much slower than in its presence, establishing the
templated nature of the three-element reaction.
To test the overall reactivity of the Sandwich probe, we
compared its reaction rate to that of a previously described,
0
single-electrophile 5 -dabsylate probe. Interestingly, the reaction
of the Sandwich probe with both nucleophiles had a similar
0
0
rate as a control 5 -single dabsylate probe with the 3 -nucleo-
phile (Fig. 2B), which is consistent with the fastest step of the
Sandwich probe having the same reactive groups as this single-
electrophile control. Notably, two reactions proceed in the
Sandwich case in the same amount of time, despite the fact
that the second set of reacting groups of the Sandwich probe
0
(
at its 3 end) are inherently less reactive.
We quantified the fluorescence signal enhancements over
0
time for the Sandwich reaction and 5 -dabsyl control.
Importantly, the results showed that the new probe yields a
substantially higher turn-on signal (48-fold at 4 h) than the
singly reactive case (32-fold) (Fig. S1B, ESIw). We also measured
signal-to-background (S/B) ratios as a function of time for the
Sandwich configuration and singly reactive control (Fig. S2,
ESIw). The new geometry yielded higher S/B (16 compared to 6)
at the maximum, apparently due to a combination of lower
initial fluorescence and reduced off-template signal due to the
presence of two quenchers/leaving groups.
Fig. 2 (A) Kinetics of Sandwich probe reactions with different
nucleophile combinations; (B) comparison of kinetics for Sandwich
and single dabsylate probe reactions with and without template;
(C) kinetics of Sandwich probe reactions on templates with single
nucleotide polymorphisms. Conditions: 100 nM quenched probe and
template, 120 nM nucleophile probes, 70 mM PIPES, 10 mM MgCl
2
,
5
0 mM DTT for 4 h at 37 1C with 494 nm excitation and 522 nm
emission. Quenched probes were added to reaction mixture after 4 min
and nucleophile probes 4 min after that.
In order to verify that the Sandwich probe undergoes two
ligation reactions, we used denaturing gel electrophoresis to
evaluate the reaction products. The reactions were run for four
hours at 37 1C, desalted, and loaded onto the gel. The
Sandwich probes were added in reactions with neither
gel was imaged by fluorescence. Results (Fig. 3) showed that
the lane containing only the Sandwich probe alone yielded one
faintly fluorescent band corresponding to the unreacted or
partially hydrolyzed probe. The control reactions containing
only one of the two nucleophiles gave two bands on the gel,
corresponding to the unligated and singly-ligated products.
0
nucleophile, both nucleophiles or only the 3 nucleophile. As
an additional control reaction, a quencher strand with only a
0
0
single 5 dabsyl was reacted with a 3 -nucleophile strand. The
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Chem. Commun., 2010, 46, 8154–8156 8155

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Fig. 3 Fluorescence PAGE gel analysis of Sandwich probe reactions.
Reactions contained 1 mM quenched probe, 1 mM template, and 1.2 mM
nucleophile in 70 mM PIPES, 10 mM MgCl , 50 mM DTT and were
2
run for 4 h at 37 1C before being desalted and loaded onto the gel.
Lane 1: Sandwich probe alone; Lane 2: Sandwich probe, template,
0
0
0
3
5
-nucleophile only; Lane 3: Sandwich probe, template, both 3 - and
0
-nucleophiles; Lane 4: Single dabsylate probe, template, 5 -nucleo-
phile only.
Fig. 4 One-step fluorescence detection of rRNA in bacterial cells with
Sandwich probes. The quenched probe (200 nM) and unlabeled helper
DNA (3 mM (see ESIw)) were incubated in 6ꢀ SSC buffer with 0.05%
This pattern is also seen for the control probe containing only
0
0
SDS at 37 1C for 2 h with E. coli cells and 2 mM of (a) both 5 - and
the 5 dabsylate electrophile.
0
0
3
-phosphorothioate nucleophile probes; (b) only 3 -phosphorothioate;
0
Finally, the reaction containing the Sandwich probe with
both nucleophiles showed three bands, corresponding to the
unligated, singly-ligated, and doubly-ligated species.
(
c) only 5 -phosphorothioate; and (d) no nucleophile. Images were
taken with a black/white camera and false-colored green.
To evaluate the selectivity of the Sandwich probe
configuration, we tested probe reactivity on synthetic DNA
targets corresponding to a site in the ribosomal RNA of
E. coli, as well as mismatches at a single position that is
known to vary in related bacteria. The central Sandwich probe
was centered on the varied nucleotide, with nucleophile probes
designed to flank this on both sides. The data show that the
reaction proceeds with high selectivity (Fig. 2C). The reaction
was run with both nucleophiles in the presence of the wild-type
template, templates with single nucleotide mismatches and
with no template. All the reactions performed with point
mutations in the template showed similar kinetics to the
reaction with no template at all. This confirms high selectivity,
and suggests that all the signals in these experiments arose
from very slow nontemplated reactions, rather than from
reaction on the mismatched template.
nontemplated signal due to the presence of two quencher
groups, yield favorable signal to background kinetics, and
can detect RNA sequences in intact bacterial cells. Surprisingly,
the requirement for two reactions does not slow the overall
turn-on of the probes. The findings suggest that increasing
1
4,15
complexity in DNA templated reaction design
may not
necessarily lead to complex outcomes, and may in fact yield
improved tools for research.
This work was supported by the U.S. National Institutes of
Health (GM068122). We thank the NSF (CHE-0639053) for
support of fluorescence instrumentation in the Department of
Chemistry. We thank S. Tabakman for help with the gel
experiments and B. Frezza for helpful discussions.
Notes and references
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2
Z. L. Pianowski and N. Winssinger, Chem. Commun., 2007,
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4 Y. Huang and J. M. Coull, J. Am. Chem. Soc., 2008, 130,
238–3239.
To assess whether the results seen in buffer could translate
to a cellular context, we tested the Sandwich probe reactivity
on a ribosomal RNA target in intact E. coli cells. As a control
for the background level of signal, the nucleophile probe or
probes were omitted from the reactions. The probes were
added to cells in the presence of 0.9 M sodium chloride and
3
3
2
3
5
6
E. Jentzsch and A. Mokhir, Inorg. Chem., 2009, 48, 9593–9595.
A. P. Silverman, E. J. Baron and E. T. Kool, ChemBioChem, 2006,
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9
0 mM sodium citrate (6ꢀ SSC) and 0.05% sodium dodecyl
sulfate to aid in permeabilization, and were incubated 2 h
without any preparation or washing steps prior to imaging
under a fluorescence microscope. The results are shown in
Fig. 4. The Sandwich probes yielded a visible green signal in
the presence of both nucleophiles, but little or no signal in the
presence of either single nucleophile. Virtually no background
signal was seen in the absence of both nucleophile probes,
revealing that two templated reactions are required to engender
this signal.
7 H. Abe and E. T. Kool, Proc. Natl. Acad. Sci. U. S. A., 2006, 103,
63–268.
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8
1
9 K. Furukawa, H. Abe, K. Hibino, Y. Sako, S. Tsuneda and Y. Ito,
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N. Winssinger, J. Am. Chem. Soc., 2009, 131, 6492–6497.
1 S. Sando and E. T. Kool, J. Am. Chem. Soc., 2002, 124, 2096–2097.
1
1
12 H. Abe and E. T. Kool, J. Am. Chem. Soc., 2004, 126,
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13 I. Le Clezio, J. M. Escudier and A. Vigroux, Org. Lett., 2003, 5, 161.
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2010, 21, 1115–1120.
Taken together, the experiments demonstrate a new and
viable architecture for nucleic acid templated reactions. The
Sandwich probes show improved initial quenching and lower
8
156 Chem. Commun., 2010, 46, 8154–8156
This journal is c The Royal Society of Chemistry 2010