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Reducing Product Inhibition in DNA-Template-Controlled Ligation Reactions.

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DNA Catalysis
DOI: 10.1002/anie.200600464
Reducing Product Inhibition in DNA-TemplateControlled Ligation Reactions**
compromising the affinity of the starting compounds for the
DNA template or the ligation rate. It has been shown recently
that the introduction of a flexible linker at the ligation site
helps to lower product inhibition.[7a] As a possible general
solution to the problem of product inhibition in DNAcontrolled ligation reactions, we propose a two-step ligation?
rearrangement sequence (Scheme 1). According to this,
Christian Dose, Simon Ficht, and Oliver Seitz*
Template-directed chemical reactions display features of
enzymatic reactions.[1] The binding of reactants allows the
alignment of reactive groups to facilitate conversions that
would proceed less efficiently when performed in the absence
of the template. Templated synthesis has frequently been
applied in nucleic acid chemistry, in the construction of
nanometer-sized architectures,[2] for the encoding of amplifiable small-molecule libraries,[3] in studies of molecular
mechanisms of evolution,[4] to release drugs,[5] and as a
diagnostic means of detecting the presence of the nucleic acid
template.[6] In contrast to enzymatic reactions, particularly
ligation reactions, templates rarely exhibit catalytic turnover
since the product usually binds the template with higher
affinity than the reactants did before ligation.[7] As a result,
stoichiometric amounts of the template are required which is
of concern in studies of chemical evolution and in strategies
for DNA/RNA detection. Here, we present a potentially
general approach to reduce product inhibition in templatecontrolled ligation reactions. We show that high turnover
numbers can be achieved by means of a ligation?rearrangement sequence in which the rearrangement is designed to
reduce the template affinity of the initially formed ligation
We have explored template-controlled ligation reactions
as one of the most powerful methods for the detection of
specific DNA sequences.[6a] Chemical ligation methods can
discriminate a particular DNA from its single-base mutant by
more than 103-fold differences in ligation rates.[6c] Conventional methods of chemical oligonucleotide ligation result in
only one product molecule per mole of target template.
However, amplification of product signals is desired when the
DNA or RNA to be detected is present at low concentration.
Usually the target segment must span at least 16 nucleotides
in order to provide a unique sequence.[8] The high number of
cooperatively formed Watson?Crick base pairs poses a
problem in the design of catalytic ligation. The challenge is
to decrease the DNA affinity of the ligation product without
[*] Dipl.-Ing. C. Dose, Dr. S. Ficht, Prof. Dr. O. Seitz
Institut f*r Chemie
Humboldt-Universit.t zu Berlin
Brook-Taylor-Strasse 2, 12489 Berlin (Germany)
Fax: (+ 49) 30-2093-7266
[**] We acknowledge support from Deutsche Forschungsgemeinschaft,
Fonds der Chemischen Industrie, and Schering AG. S.F. is grateful
for a fellowship from the Grand Duchy of Luxembourg.
Supporting information for this article is available on the WWW
under or from the author.
Angew. Chem. Int. Ed. 2006, 45, 5369 ?5373
Scheme 1. General scheme of ligation?rearrangement reactions for
reducing product inhibition in template-controlled ligations.
hybridization would trigger formation of ligation intermediate 1� We envisioned a subsequent spontaneous rearrangement, which would alter the chain length of the backbone to
reduce the template affinity of the rearranged product 5. As a
result, dissociation of product?template duplex 1�would be
facilitated to liberate template 1 for a further catalytic cycle.
To enhance sequence fidelity and as additional means of
reducing the template affinity of the ligation product, we
planned to perform the ligation opposite to an unpaired
We have recently introduced native chemical peptide
nucleic acid (PNA) ligation,[6b] an extremely rapid and
selective reaction[9] that proceeds through a ligation?rearrangement sequence. The design of a ligation architecture
that would facilitate dissociation of the product?template
duplex was guided by the studies of Egholm, Buchardt, et al.,
who showed that extension of the aminoethylglycine backbone in PNA by one CH2 group resulted in destabilization of
PNA?DNA duplexes.[10] We assumed that the correct interbase distance would be required not only in seamless base
pairing (6 s bonds) but also in hybridizations involving an
abasic site (12 s bonds). Indeed, the TM = 58 8C of a PNA?
DNA duplex containing a glycine?glycine unit (interbase
distance: 12 s bonds) as an abasic-site analogue was found to
exceed the TM = 55 8C measured for a PNA?DNA duplex
containing glycine?b-alanine (interbase distance: 13 s bonds)
(Table S1 in the Supporting Information).
In the previously reported native chemical PNA ligation
of PNA?glycine thioester 2 with Cys-PNA 3 a, the thioester
intermediate 4 a is formed (Scheme 2).[6b] A subsequent S!N
acyl shift leads to contraction of the main chain which is
expected to align the nucleobases in product 5 a with an
interbase distance of 12 s bonds, perhaps more favorably than
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Scheme 2. Cys- and iCys-mediated chemical PNA ligation on DNA
templates. (R = Bn or (CH2)2SO3Na).
in thioester intermediate 4 a (interbase distance: 13 s bonds).
To reduce product inhibition, the reaction of thioester 2 with
isocysteine(iCys)-PNA 3 b was considered. In iCys-PNA 3 b
the positions of the thiol and the amino groups are
exchanged.[11] As a result, isocysteine-mediated ligation
proceeds by main-chain extension. Here the final product
5 b has an interbase distance of 13 s bonds and was hence
assumed to bind the DNA template less tightly than thioester
intermediate 4 b.
The concept was first evaluated by estimating the affinity
of PNA probes targeted against the carcinogenic G12V
mutation of a 16mer ras gene segment. Melting-temperature
analysis confirmed that PNA?DNA duplexes containing the
final product 5 b of iCys-mediated ligation (TM = 53 8C) are
less stable than duplexes that contain the product 5 a of
cysteine native chemical ligation (TM = 57 8C). Next studied
was the template-independent reaction of Cys-PNA 3 a and
iCys-PNA 3 b with PNA-glycine thioester 2 under the
conditions of bimolecular native chemical ligation. At
100 mm concentration both Cys- and iCys-mediated ligations
proceeded smoothly furnishing product 5 a in 77 % yield and
5 b in 49 % yield, respectively, after 6.5 h (Figure 1 a).
Comparison of the initial rates revealed that templateindependent bimolecular iCys-mediated PNA ligation is 4.4
times slower than the corresponding Cys-mediated reaction.
At a probe concentration of 1 mm, bimolecular ligation was
extremely slow (Table S3 in the Supporting Information).
Interestingly, Cys and iCys ligations were equally fast when
performed on the matched DNA template RasT (Figure 1 b).
In both cases addition of 1 mm template RasT led to a
Figure 1. Time courses of the Cys- (&) and iCys-mediated (*) PNA
ligation for a) 100 mm 2 and 3 (100 mm Na2HPO4, sat. BnSH, pH 7.4,
25 8C) and b) at 1 mm 2 and 3 on matched DNA RasT or single-basemismatched DNA RasG (^, only iCys-mediated ligation shown, 10 mm
Na2HPO4, 10 mm NaCl, sat. BnSH, pH 7.4, 25 8C).
dramatic rate enhancement: both 5 a and 5 b were formed in
20 % yield after only 90 s reaction time (apparent secondorder rate constant: kapp = 2.470 m 1 s1). Even higher rate
accelerations were obtained with mercaptoethanesulfonate
(kapp = 10.820 m 1 s1), leading to a 75 % yield of ligation
product after a reaction time of 5 min (data not shown). Thus,
the rate of the iCys- and Cys-mediated PNA ligation is
significantly higher than that of alternative chemical reactions
for which sequence specificity has been demonstrated[6d?i] and
is in the range of the fastest DNA-templated ligations.[12]
The iCys ligation was found to proceed with the same high
sequence fidelity (the initial rate on single-base-mismatched
DNA RasG was 3450-fold slower than on RasT) as Cys
ligation (Table S2 in the Supporting Information).[6c] It can be
concluded that the use of iCys rather than Cys in native
chemical PNA ligation reduced the rate of the off-template
background reaction without affecting speed and sequence
selectivity of the DNA-templated reaction.
The above-discussed features of iCys-mediated ligation
were considered to be useful for achieving catalytic turnover.
The turnover numbers (TONs) were determined by measuring Cys and iCys ligation at different template/probe ratios.
Background signals from reactions in absence of DNA target
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 5369 ?5373
RasT were subtracted in order to ensure that the determined
yields were only a result of template-dependent reactions. It
was expected that a large excess of ligation probes relative to
the template would facilitate the displacement of the ligation
product. Indeed, Figure 2 shows that significant turnover
Figure 2. Turnover numbers in Cys- (white bars) and iCys-mediated
(black bars) PNA ligations (10 mm Na2HPO4, 150 mm NaCl, 10 mm
mercaptoethanesulfonic acid (MESNA), pH 7.4, 25 8C, 24 h).
numbers were obtained at low template/probe ratios. For
example, at a probe concentration of 1 mm and DNA
concentration of 10 nm, iCys ligation yielded a turnover
number of 3.5 which improved to 43 for a target concentration of 0.1 nm. Increases of the ligation probe concentration led to further improvements of turnover numbers at any
given template/probe ratio. For example, signal amplification
in Cys ligation reached 14-fold at 1 mm and 42- and 51-fold at
probe concentrations of 5 mm and 10 mm, respectively. However, most noticeable was the observation that in all cases the
iCys-mediated chemical ligation provided higher turnover
numbers than the Cys-mediated reaction. The highest signal
amplification was observed for the iCys ligation with 226
turnovers at a ligation probe concentration of 10 mm and a
Angew. Chem. Int. Ed. 2006, 45, 5369 ?5373
DNA target concentration of 1 nm ; this exceeds turnover
numbers reported for alternative methods (vide infra).[7]
The replacement of Cys by iCys has no effect on the
stability of probe稤NA complexes; both duplexes 3 a稲asT
and 3 b稲asT have a melting temperature of 38 8C. Thus, the
remarkable improvement of the turnover numbers observed
in isocysteine ligation must be the sole result of the subtle
changes of the ligation architecture. The reduced rate of the
bimolecular iCys ligation can be explained by the reduced
nucleophilicity of the iCys thiol relative to that of the Cys
thiol. However, the equal reaction rates of DNA-controlled
iCys and Cys ligation suggest that template effects can
compensate for the attenuated reactivity of iCys.[13] As a
result, DNA-triggered iCys ligation is 43 000 times faster than
the template-independent reaction, while Cys ligation features rate accelerations of 10 000-fold rate accelerations
(Table S3 in the Supporting Information). Furthermore, the
lower DNA affinity of the iCys-ligation product suggests that
the increased flexibility that is provided by a rearrangement
that proceeds by main-chain extension helps in facilitating
In the described work, product signals were quantified by
high-pressure liquid chromatography which facilitated the
careful analysis of reaction pathways. As fluorescence-based
methods enable real-time measurements, they have advantages in terms of ease and speed of detection.[6f] We reckoned
that an approach based on fluorescence resonance energy
transfer (FRET) between a 6-carboxyfluorescein (FAM)
fluorophore in the PNA-glycine thioester 6 and a 5-carboxytetramethylrhodamine (TAMRA) unit in iCys-PNA 7 would
enable the specific detection of formed product.[14] HPLC
analysis revealed that after 60 min the ligation had proceeded
with 52 % yield on the matched DNA Ras2 T and less than
1 % yield on the single-base-mismatched template Ras2 G
(Figure S5 in the Supporting Information). The same ligation
reaction was analyzed in real time by fluorescence spectroscopy (excitation at 470 nm). Figure 3 a shows that the
fluorescence spectrum of probes 6 and 7 in the absence of
template is dominated by FAM emission at 523 nm. The
addition of template Ras2 T triggered ligation and resulted in
a marked change of the fluorescence spectrum characterized
by an increase of TAMRA emission (585 nm) and a decrease
of FAM emission (523 nm). Melting-temperature analysis for
the duplexes 6稲as2 T (TM = 38 8C) and 7稲as2 T (TM = 36 8C)
confirmed that both PNA probes were aligned on the
template at 25 8C. It is noteworthy that adjacent hybridization
of two probes that are unable to ligate proved considerably
less efficient in inducing fluorescence changes (Figure 3 a).
Product formation can be monitored by means of the ratio of
fluorescence intensities F585/F523 or by directly following
TAMRA emission at 585 nm (Figure 3 b,c). Either method
generates a positive signal and, in contrast to other methods
that rely on quenchers as leaving groups, is unaffected by
hydrolysis. The sequence selectivity of the fluorescencemonitored ligation was high. A comparison of the initial
rates, determined by analysis of the increase of F585/F523,
revealed that at 25 8C the reaction proceeded 127 times faster
on matched DNA Ras2 T than on mismatched DNA Ras2 G.
Perfect selectivity was obtained at 37 8C. Monitoring of
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
not described. Kool et al. introduced a ligation format
wherein single-base-mismatched probes coupled 180 times
slower than matched probes.[6f] The incorporation of a flexible
linker at the ligation site resulted in 12-fold sequence
selectivity and 92 turnovers under turnover conditions.[7a]
The use of a fluorescence quencher as a leaving group
allowed fluorescence-based detection of signals even within
living cells. However, fluorescence signaling is not specific for
product formation and can arise also from off-target hydrolysis reactions. KrHmer, Mokhir, et al. designed a system in
which adjacent hybridization of two PNA probes, one bearing
a copper complex and the other bearing an ester group,
triggered ester hydrolysis. This reaction featured 500-fold rate
accelerations on DNA template, 102-fold single-nucleotide
specificity, and a TON of 5.[6i] Taylor et al. showed the
potential for signal amplification in elegant PNA-based
systems, in which adjacent hybridization of imidazole- or
azide-containing probes prompted deacylation of acylquenched fluorogenic probes.[16] Fluorescence signaling was
not specific for product formation, and the template-controlled deacylation occurred 36 times faster than the offtemplate reaction.
In conclusion, we have introduced a new PNA ligation
reaction, native chemical isocysteine ligation, and showed
significant enhancements of signal amplification over methods based on cysteine ligation and alternative DNA ligation
methods. The results demonstrate, for the first time, that the
involvement of a flexibility-increasing rearrangement step
can reduce product inhibition in template-controlled ligation
Figure 3. Fluorescence monitoring of the iCys-mediated PNA ligation
of PNA probes 6 and 7. a) Fluorescence spectra before addition of
matched DNA Ras2 T (c) and 60 min after addition (a). The
spectrum of Acctcttc(FAM)cccacGly-OH 9 and 7 after 60 min incubation with Ras2 T is also shown (g). b) Ratio between fluorescence
intensities at 585 nm and 523 nm measured for ligations at 25 8C and
c) fluorescence intensity at 585 nm for ligations at 37 8C on matched
DNA Ras2 T (c) and mismatched DNA Ras2 G (a). (1 mm
probes, 10 mm Na2HPO4, 150 mm NaCl, 10 mm MESNA, pH 7.4,
R = (CH2)2SO3Na).
TAMRA emission showed that ligation was not detectable on
mismatched template Ras2 G (Figure 3 c).
The presented data demonstrated high single-nucleotide
selectivity (3450:1), speed (kapp = 10.820 m 1 s1), and rate
acceleration (43 000-fold) of DNA-template-triggered iCys
ligation. We also showed signal amplification (TON = 226)
and a convenient means of signal detection by fluorescence
spectroscopy. Strategies to reduce product inhibition in
template-controlled oligonucleotide reactions have been
reported. Kiedrowski et al. introduced a surface-promoted
amplification protocol which unlike the present method
required successive denaturation?annealing cycles.[15] Albagli
et al. used a photoinduced [2+2] cycloaddition for selfreplication of oligonucleotide systems.[7c] Lynn and Zhan
proposed a DNA ligation of trinucleotides that proceeds by
reductive amination and resulted in more than 50 turnovers.[7b] Sequence specificity and fluorescence read-out were
Received: February 3, 2006
Published online: July 17, 2006
Keywords: FRET (fluorescence resonance energy transfer) �
native chemical ligation � sequence analysis � single-nucleotide
polymorphism � template synthesis
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reducing, reaction, ligation, inhibition, dna, controller, product, template
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