Amplified Signaling and Sequence Selectivity of Nucleic Acid Templated Reactions
FULL PAPER
range of the transfer rate observed for 7c+12 (Table 1).
With one equivalent (200 nm) probes, the rate of pyrene
provements in signaling rates observed when the probe con-
centration was decreased (at constant template concentra-
tion) suggest that many previous reactions may have been
performed at conditions that provided high levels of back-
ground reaction.[4,16,36] Of note, the reduction of probe con-
centration increases the contribution of the templated reac-
tion and, thereby, increases sensitivity.
The results furthermore demonstrate that the selection of
an appropriate readout method can improve selective signal-
ing. We introduced the excimer formation triggered by
pyrene transfer as a method that allows the specific detec-
tion of the transfer product only. We showed that measure-
ments of excimer fluorescence provided up to fourfold in-
creases in the signaling rates compared with the previously
described transfer of a fluorescence quencher from a fluo-
rescein- to a rhodamine-modified probe.
Standard methods for the sequence-selective detection of
nucleic acids often employ the use of hybridization probes
such as molecular beacons (MB)[29–31] or adjacent probes.[37]
These approaches do not enable signal amplification. For
highly sensitive detection, hybridization probes are applied
in combination with the polymerase chain reaction (PCR).
Compared with probes used for target-catalyzed reactions,
hybridization probes are more readily accessible and the
presence of the target can be detected within a shorter re-
sponse time. On the other hand, the high sensitivity of
target-catalyzed reactions certainly is a valuable asset, which
might facilitate the detection of low abundant nucleic acid
material as required in PCR-less detection of nucleic acid
targets[21] and live-cell RNA analysis.[5]
transfer was 44-fold higher on matched target RasT (v1RasT
=
157 pmsꢀ1) than on mismatched RasG (v1RasG =3.5 pmsꢀ1).
Substoichiometric amounts of RasT also proved efficient in
furnishing rapid enhancements of the excimer signal
(v0.1RasT =17.2 pmsꢀ1). In the presence of 0.1 equivalents
(20 nm) of target DNA, the selectivity increased to s=67.
This exceeds the selectivity obtained in the reaction of 7c
with 12 (Figure 2b). Figure 5b compares the relative slopes
of the signal achieved upon excimer formation (14+15,
black) with the one determined by monitoring the FFAM
/
FTMR ratio (Dabcyl transfer: 7c+12, white). The presence of
0.1 equivalents of RasT accelerated the excimer signaling by
a factor of 140, which exceeds the 98-fold acceleration of
the FFAM/FTMR signal. The addition of one equivalent of
RasT triggers higher accelerations of signaling rates. Under
these conditions, excimer signaling surpassed FFAM/FTMR sig-
naling by a factor of four. The DNA-catalyzed transfer of a
pyrene reporter is the first transfer reaction that triggers the
activation of a fluorescence signal that is insensitive to non-
specific background hydrolysis. Therefore, the background
signal is lowered significantly.
Conclusions
We used the transfer of a reporter group, a reaction that fol-
lows the mechanism of the native chemical ligation, as a
model system for a DNA-templated reaction. We demon-
strated the influence of important reaction parameters such
as a) reactivity of functional groups, b) affinity of probes for
the target, and c) the readout system on sensitivity and se-
lectivity of the DNA-catalyzed reaction. For the modulation
of the thioester reactivity, fluorescence-based real-time
measurements exposed a general trend. Decreases in the re-
action rate on the target resulted in increased sequence se-
lectivity. Based on these experiments, we identified special-
ized probe sets that either provide very high turnover num-
bers or very high sequence selectivity.
A more elaborated readout system, in which transfer of
the Dabcyl quencher triggered the activation of fluorescein
fluorescence and the quenching of rhodamine fluorescence,
allowed a clear distinction between selective transfer reac-
tions and unselective hydrolysis reactions. We used this
system to assess how changes of the affinity of the probes
for the template influence the template-catalyzed reaction.
The probe affinity was conveniently adjusted through
changes in the reaction temperature. The template-catalyzed
reaction proceeded at the highest rates close to the melting
temperature of the probe–template duplexes. We assume
that the optimum conditions between sufficient template af-
finity of the probes and effective strand exchange lie close
to the melting temperature of the formed duplexes. Our in-
vestigations revealed that the signaling power of a given
probe set can be improved not only through reaction design
but also through adjustment of probe concentration. The im-
Experimental Section
Materials and instruments: PNA monomers were purchased from Ap-
plied Biosystems. DNA was purchased from BioTeZ Berlin Buch GmbH,
Germany, in HPLC quality. Water was purified with a Milli-Q ultra pure
water purification system. Automated linear solid-phase synthesis was
performed by using an Intavis ResPep parallel synthesizer equipped with
micro scale columns for PNA synthesis. For details of probe synthesis,
see the Supporting Information. Analytical HPLC was performed with a
Merck-Hitachi Elite LaChrom chromatograph (column: Varian Polaris
C18 A 5 m 250ꢃ46, pore size 220 ꢄ) at 558C. Eluents: A (98.9% H2O,
1% acetonitrile, 0.1% trifuloroacetic acid (TFA)) and B (98.9% acetoni-
trile, 1% H2O, 0.1% TFA) were used in a linear gradient with a flow
rate of 1 mLminꢀ1 for analytical and 6 mLminꢀ1 for semi-preparative
HPLC. For probe characterization, MALDI-TOF mass spectra were re-
corded with a Voyager-DE Pro Biospectrometry workstation of PerSep-
tive Biosystems.
Fluorescence-based kinetic measurements: Fluorescence spectroscopy
was performed by using a Varian Cary Eclipse spectrometer. Measure-
ments were carried out in fluorescence quartz cuvettes (4ꢃ10 mm). The
buffer solution (10 mm NaH2PO4, 100 or 200 mm NaCl, and 0.2–2 mm tris-
carboxyethylphosphine) was degassed and the pH value adjusted to 7. To
prevent adsorption of TMR- and Py-labeled probes on the glass surface,
roche blocking reagent (0.1 gLꢀ1) was added. Subsequent manipulations
were carried out while avoiding unnecessary exposure to oxygen. The
buffer solution (final volume 1 mL) was placed in a cuvette and accepting
probe (8, 12 or 15; 0.02–0.3 nmol) and DNA (RasT or RasG; if required)
were added. After setting the solution to the required temperature, do-
nating probe (7a–g or 14; 0.02–0.2 nmol) were added and the cuvette was
Chem. Eur. J. 2009, 15, 6723 – 6730
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
6729