DNA-catalyzed transfer of a reporter group

Article abstract of DOI:10.1021/ja0670097

Herein we describe a novel DNA-catalyzed transfer of a reporter group from a donating to an accepting oligo-peptide nucleic acid (PNA), generating high catalytic turnover numbers in a DNA-sequence specific manner. The demonstrated transfer of the Dabcyl g

Full text of DOI:10.1021/ja0670097

Published on Web 11/16/2006
DNA-Catalyzed Transfer of a Reporter Group
Tom N. Grossmann and Oliver Seitz*
Institut fu¨r Chemie, Humboldt-UniVersita¨t zu Berlin, Brook-Taylor-Strasse 2, D-12489 Berlin, Germany
Received September 29, 2006; E-mail: oliver.seitz@chemie.hu-berlin.de
DNA-templated reactions are emerging as a general approach
to control the reactivity of synthetic molecules by modulating the
effective molarity.1 DNA templates align the reactive groups of
these molecules to allow fast and selective reactions at reactant
concentrations that are much lower than those required for
conventional synthesis. Among various types of chemistries, most
DNA-directed reactions involve the covalent ligation of two
modified oligonucleotide strands.2 In general, ligation reactions
suffer from an increased affinity of the product to the DNA
template. This causes product inhibition and prevents high catalytic
activity of the nucleic-acid template. However, catalysis would be
a valuable asset, particularly in DNA and RNA detection chemis-
tries. Catalytic turnover would provide for signal amplification and
would, thus, enable highly sensitive DNA and RNA detection in
homogeneous solution and in living cells. To reduce product
inhibition, flexibility enhancing ligation reactions have been
developed.2i,m However, ligation products still have higher template
affinity than the reactant probes. Other approaches to prevent
product inhibition involve DNA-directed O-deacylation of modified
oligonucleotides by ester hydrolysis3 or Staudinger reaction.4 Even
though these approaches yield product complexes with unchanged
stability, they have not led to a large number of catalytic turnover
so far. In this Communication we show that DNA-directed transfer
reactions may present a general solution to the problem of product
inhibition as evidenced by high turnover (TO) and signal amplifica-
tion.
Our design5 of a reaction in which the DNA analyte acts as
Figure 1. (top) Catalytic cycle of the DNA-catalyzed transfer of a reporter
catalyst involves an iso-cysteine(iCys)-mediated transfer of a
reporter group (Figure 1). A donating 1 and accepting 2 peptide
nucleic acid (PNA) probe are designed to hybridize adjacently to
the complementary DNA template RasT. The reporter group (dark
red) in 1 is covalently bound to a PNA-peptide conjugate (orange)
as thioester. Probe 2 (blue) bears an N-terminal iCys capable of
attacking the thioester in probe 1 in a native chemical ligation-like
fashion.2m,6 Adjacent hybridization of probe 1 and 2 in ternary
complex RasT‚1‚2 triggers transthioesterification to form 4*. This
is followed by an irreversible SfN-acyl migration, yielding ternary
complex RasT‚3‚4. As result, the reporter group is transferred from
PNA 1 to PNA 2. Catalysis requires that products 3 and 4 in ternary
complex RasT‚3‚4 can be replaced by nonreacted probes 1 and 2.
Reactant and product probes have been fashioned to have similar
affinity to DNA RasT and, thus, strand exchange may proceed
quickly.
We decided to develop a fluorescence-based read-out, which
facilitates real-time measurements. Probe 1 was N-terminally
modified with 6-carboxyfluorescein (FAM, orange) and 2 was
labeled with 5-carboxytetramethylrhodamine (TAMRA, blue) on
the R-amino group of a C-terminal lysine. In both cases [2-(2-
aminoethoxy)ethoxy]acetic acid (AEEA) was used as spacer to
minimize the influence of the fluorophores on the hybridization
properties. 4-[4-Dimethylamino)phenylazo]benzoylglycine (Dabcyl-
Gly), representing the reporter group (dark red), was attached as
group (dark red) from the donating 1 to the accepting PNA probe 2; (bottom)
iCys-mediated transfer reaction.
Figure 2. (a) FAM (λex ) 465 nm, λem ) 525 nm) and TAMRA (λex
)
558 nm, λem ) 593 nm) fluorescence of the DNA-directed transfer reaction
(probe 1 and 2) in the presence of 1 equiv RasT; (b) time courses of relative
FFAM/FTAMRA ratio at different RasT and RasG concentrations (200 nM
probes, 10 mM NaH2PO4, 200 mM NaCl, 0.2 mM TCEP, 0.1 mg/mL roche
blocking reagent, pH 7.0, 37 °C).
thioester to probe 1. The Dabcyl group is capable of quenching
both FAM and TAMRA fluorescence when in proximity.
Figure 2a shows the time-dependent intensities of FAM (orange
line) and TAMRA (blue line) fluorescence, in a reaction of probe
1 and 2 in the presence of 1 equiv matched DNA RasT. Prior to
transfer the FAM fluorescence of probe 1 is quenched by Dabcyl.
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10.1021/ja0670097 CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Useful 7-fold (after 5 h) or 3-fold (after 7 h) enhancements were
obtained in the presence of 0.01 equiv (1 nM) and 0.001 equiv
(0.1 nM) DNA, respectively. At 0.1 nM RasT FFAM/FTAMRA
signaling rate is 2-fold over background. Lower DNA template
concentrations would require the use of longer transfer probes (>10
nt) to increase the template affinity. The predominant source of
background is thioester hydrolysis (see Supporting Information).
The use of less reactive thioesters and/or the transfer of fluorescent
reporter groups should allow for further reductions of background.
Previous nonenzymatic DNA-catalyzed reactions based on liga-
tion required high probe concentrations (10 µM) and large probe
excess (10.000-fold) to drive displacement of the product from the
product-template duplex, at the cost of low ligation rate and high
ligation background.2i,m In addition, amplification of a fluorescence
signal based on ligation has not been demonstrated. The potential
for signal amplification has been shown in DNA-triggered cleavage
reactions of esters in PNA-probes.3 In these cases, turnover numbers
were lower than 10, presumably because of relatively fast off-
template reactions.
The DNA-directed transfer7 of a reporter group developed by
us is new and is the first reaction to combine high catalytic activity
of the DNA analyte with useful reaction yields and low background
under turnover conditions. The described Dabcyl transfer is restoring
fluorescein emission while switching off rhodamine emission. This
allowed monitoring of both transfer (FAM + TAMRA) and
hydrolysis (FAM). Other reporter groups are possible as we expect
a general applicability of the new transfer reaction in DNA catalysis.
Future studies will be aimed at increasing signal output by applying
this concept to other types of reactions, oligonucleotides, and read-
out systems.
Figure 3. Transfer reaction of probe 1 and 2: (a) transfer yields after 24
h and turnover numbers (TO); (b) time courses of relative FFAM/FTAMRA
ratio at shown RasT concentrations (100 nM probes, 10 mM NaH2PO4,
200 mM NaCl, 1 mM TCEP, 0.1 mg/mL roche blocking reagent, pH 7.0,
32 °C).
Dabcyl-Gly transfer and formation of probe 3 results in an increase
of FAM fluorescence. Simultaneously, the fluorescence intensity
of TAMRA decreases, owing to the transfer of Dabcyl-Gly to probe
2. This setup allows the specific detection of product formation.
The transfer reaction can be further monitored by means of the
ratio of fluorescence intensities FFAM/FTAMRA (Figure 2b). The time
course of relative FFAM/FTAMRA ratio shows a rapid signal increase
in the presence of 1 equiv matched DNA RasT (red line) reaching
17-fold enhancement after 90 min. Compared to the reaction in
the absence of DNA (black line), the transfer reaction is accelerated
by a factor of 1720. The useful 5-fold increase above background
signal level is obtained already after 3.5 min. Substoichiometric
amounts of RasT (0.1 equiv DNA, yellow line) proved also efficient
in conferring a rapid signal increase and provided 7.0 TO after 1
h. The initial rates on single-base mismatched DNA RasG (dashed
lines) are significantly reduced. At 200 nM probes and 20 nM target
DNA, the transfer reaction on the mismatched RasG proceeds 44
times slower than on RasT.
Acknowledgment. We acknowledge support from Schering AG.
T.N.G. is grateful for a fellowship from the Studienstiftung des
deutschen Volkes.
The next aim was to specify the catalytic activity of the DNA
template. With a load of 0.01 equiv (1 nM) DNA catalyst RasT,
the transfer reaction furnished 69% of transfer product after 24 h
(Figure 3a). In the absence of DNA, 100 nM probe 1 and 2 yielded
3.4% background transfer. It was concluded that 0.01 equiv DNA
RasT conferred 66 TO. After 12 h we already observed 54 TO
(see Supporting Information). A notable 25% yield (215 TO) was
obtained with 0.001 equiv RasT (0.1 nM). Ligation reactions require
100 times higher DNA concentrations and 10 times more probe
excess to obtain similar TO.2i,m The highest turnover number of
402 was achieved with 0.0001 equiv RasT (0.01 nM), surpassing
TO reported for alternative sequence-specific methods.2d,4 It has
often been observed that the speed of the DNA-catalyzed reaction
is reduced at very low DNA/probe ratios to an extent that reaction
yields are dominated by the off-template background reaction.
However, the 7.4% transfer product 4 obtained with 0.0001 equiv
RasT (0.01 nM) demonstrate that the DNA-catalyzed transfer
(4.0%) proceeds more efficiently than the background transfer
(3.4%), a result not being reported yet.
The catalytic activity of the DNA analyte was considered useful
for achieving signal amplification in fluorescence-based DNA
detection. It is instructive to estimate fluorescence signaling
provided by a conventional hybridization probe used in homoge-
neous DNA detection. A hybridization probe that yields 17-fold
fluorescence enhancement upon quantitative hybridization will give
only 2.6, 1.16, or 1.016-fold increases in the presence of 0.1, 0.01,
and 0.001 equiv DNA, respectively. In contrast, our transfer reaction
catalyzed by 0.1 equiv DNA (10 nM) provides for 10-fold
enhancement of the FFAM/FTAMRA signal within 1 h (Figure 3b).
Supporting Information Available: Synthesis, experimental de-
tails, and HPLC data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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