DNA with 3′-5′-disulfide links - Rapid chemical ligation through isosteric replacement

Article abstract of DOI:10.1002/anie.201310644

Efforts to chemically ligate oligonucleotides, without resorting to biochemical enzymes, have led to a multitude of synthetic analogues, and have extended oligomer ligation to reactions of novel oligonucleotides, peptides, and hybrids such as PNA.1 Key re

Full text of DOI:10.1002/anie.201310644

Angewandte
Chemie
DOI: 10.1002/anie.201310644
Chemical DNA Ligation
DNA with 3’-5’-Disulfide Links—Rapid Chemical Ligation through
Isosteric Replacement**
Volker Patzke,* John S. McCaskill, and Gꢀnter von Kiedrowski
Abstract: Efforts to chemically ligate oligonucleotides, without
resorting to biochemical enzymes, have led to a multitude of
synthetic analogues, and have extended oligomer ligation to
reactions of novel oligonucleotides, peptides, and hybrids such
as PNA.[1] Key requirements for potential diagnostic tools not
based on PCR include a fast templated chemical DNA ligation
method that exhibits high pairing selectivity, and a sensitive
detection method. Here we report on a solid-phase synthesis of
oligonucleotides containing 5’- or 3’-mercapto-dideoxynucleo-
tides and their chemical ligations, yielding 3’-5’-disulfide bonds
as a replacement for 3’-5’-phosphodiester units. Employing
a system designed for fluorescence monitoring, we demonstrate
one of the fastest ligation reactions with half-lives on the order
of seconds. The nontemplated ligation reaction is efficiently
suppressed by the choice of DNA modification and the 3’-5’
orientation of the activation site. The influence of temperature
on the templated reaction is shown.
and routinely used to immobilize DNA on gold surfaces[14] or
on thiolated supports.[15] Disulfide-based crosslinking by
means of thiol-modified bases can be used to transform
double-stranded DNA into covalently linked species.[16]
Disulfide-based templated ligations were developed with
phosphorothioates as functional groups.[17] These systems
yielded significant amounts of side products through non-
templated reactions. Ligations based on the formation of 3’-5’-
disulfide linkages were suggested by Schwartz and Orgel as
early as 1985.[18] Witch and Cosstick synthesized dithymidine
building blocks with 3’-5’-disulfide linkages, aiming to use the
latter to generate a novel class of antisense ONs.[19]
Molecular modeling suggested that the replacement of the
phosphodiester bridge by a 3’-5’-disulfide bond expressing
a favored 908 dihedral angle should result in only little
conformational distortion of the B-DNA backbone
(Figure 1). Our approach involves the formation of a 3’-5’-
disulfide bond through a disulfide-exchange reaction, which is
P
revious studies on template-directed reactions[2] and self-
replicating systems[3] based on chemical ligations[4] employed
the formation of phosphodiester, pyrophosphate, and phos-
phoramidate bonds through carbodiimide or imidazolide
activation. Phosphoramidate bond formation has been devel-
oped further to yield highly efficient and fast templated
polymerization syntheses.[5] Several chemical methods based
on the autoligation of oligonucleotides for diagnostics are
based on phosphorothioate with iodide or tosylate,[6] Diels–
Alder, and click reactions.[7] Furthermore, many template-
based methods for nucleic acid detection have been reported
based upon the Staudinger reaction,[8] native chemical
ligation,[9] metal-catalyzed reactions,[10] photocatalyzed reac-
tions,[11] and nucleophilic aromatic substitutions.[12] The tem-
plate approach has resulted in rate accelerations of up to
1000-fold and half-lives as low as 90 s.[13]
Figure 1. Overlay of two geometry-optimized models for B-DNA (com-
putation: semiempirical, PM3; counterstrand removed), one contain-
ing a 3’-5’-phosphodiester, the other a 3’-5’-disulfide linkage. The C(3’)-
S-S-C(5’) dihedral angle is close to the optimum of 908.
Thiol modifications are widely used in oligonucleotide
(ON) synthesis. Thiol modifiers are commercially available
[*] Dr. V. Patzke, Prof. Dr. G. von Kiedrowski
Lehrstuhl fꢀr Bioorganische Chemie, Ruhr-Universitꢁt Bochum
44780 Bochum (Germany)
attractive because it is fast and highly selective, and can be
directed by control of the redox conditions and pH. The
structural similarity between the disulfide bond and the native
phosphodiester bond should favor the templated reaction
pathway, whereas the application of longer and flexible
ligation sites resulted in increased turnover rates.[6b]
E-mail: volker.patzke@rub.de
Prof. Dr. J. S. McCaskill
BioMIP: Microsystems Chemistry and BioIT
Ruhr-Universitꢁt Bochum
44780 Bochum (Germany)
In earlier phosphoramidate replication studies, the CG
motif was found to be the most effective ligation site because
of base-stacking effects.[20] We synthesized DMT- and base-
protected thiol-deoxynucleosides 5’-HS-dC and 3’-HS-dG
and immobilized them as disulfides on a thiolated controlled
pore glass (CPG) solid support. The thiol-ONs were prepared
[**] This research was funded by the European Community’s Seventh
Framework Programme (FP7/2007-2013) under grant agreement
no. 222422 (“ECCell—Electronic Chemical Cell”, a project of the EU
FP7-IST-FET Open Initiative).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201310644.
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from the corresponding 3’- and 5’-phosphoramidites using the
solid supports as shown in Figure 2 (for details see the
Supporting Information). The solid supports allow the
utilization of standard synthesis protocols.
micromolar concentrations of thiolated DNA precursors to
minimize spontaneous nontemplated ligation. Sequences with
9-mer precursor molecules and 18-mer templates were chosen
for the ligation to provide a sufficient population of the
termolecular complex at such low concentrations. In the 9–18-
mer system, two complementary 3’-S-ON, 5’-S-ON pairs and
the corresponding templates were synthesized (Table 1). The
6-FAM-labeled ONs were converted into activated and
quenched precursors using a 20-fold excess of the dabcyl
linker molecule (pH 7.0 buffer, RT, 15 min).
Table 1: Oligonucleotide sequences and temperature effects for non-
Figure 2. Structure of the solid supports employed in the synthesis of
5’- and 3’-thiol-ONs (details in the Supporting Information). DMT:
dimethoxytrityl, iBuG: N2-isobutyrylguanine, BzC: N4-benzoylcytosine.
templated ligation experiments.
Ligation
3’-Activation
5’-Activation
3’-S-ON
5’-S-ON
FATTATACCGQ
A
TACTTCAAGS E
QCGGTATAATF D
SCTTGAAGTA B
We chose Ellmanꢀs reagent to preactivate one precursor
molecule as a mixed disulfide. A further customized modifi-
cation could be introduced through the carboxyl moiety of
Ellmanꢀs reagent: a dabcyl unit as a quencher leaving group
for FRET detection. This feature, combined with a 6-FAM-
labeled ON, which is common in existing probes,[6b–d] made it
possible to monitor the reaction directly by fluorescence
down to nanomolar concentrations (Scheme 1B; for details
see the Supporting Information). The quencher can be
introduced to the thiol-ON at either the 3’- or 5’-end.
T [8C]
k1 [mÀ1 sÀ1
]
35
38
40
45
55
33
37
43
54
62
391
547
718
844
1413
Ligation reactions were carried out to investigate the
nontemplated background reaction (for details see the
Supporting Information). Surprisingly, the 3’- and the 5’-
electrophiles underwent the nontemplated background reac-
tions at very different rates. For a 3’-electrophile (A) and a 5’-
nucleophile (B) we observed significantly lower rates than
with a 5’-electrophile (D) and a 3’-nucleophile (E) (Table 1).
The nontemplated reactions show conversions at pH 7 (on
a timescale of 30 min) of 6–11% for the 3’-activation versus
60–90% for 5’-activation. These results cannot be explained
by nucleophilicity, since the 5’-thioate is expected to be the
stronger nucleophile. Experiments with interchanged nucle-
ophiles, leading to 3’-3’ and 5’-5’ ligations, also gave relatively
low yields with the 3’-electrophile (see data in the Supporting
Information). The effect apparently originates from steric
hindrance. 3’-Activation (see Table 1) showed only minor
temperature dependence (over this range). A template effect
was observed for both activation pathways, but for the 5’-
activation the nontemplated reaction is dominant. Conse-
quently, we restricted our examination of template and
temperature effects to the 3’-activated system.
On ligation with a thiol-ON as a nucleophile, the quencher
(Q) is liberated producing a fluorescence signal (Sche-
me 1A). Preliminary ligation experiments with 5’-activation
had revealed a very fast ligation reaction,[21] mandating low
To prove that the products are pure and that the ligations
are unambiguous we carried out qualitative gel analyses of the
templated ligation reactions (Figure 3). The two fluorescent
bands were identified as the precursor molecule and the
product by MALDI-TOF mass spectrometry (see the Sup-
porting Information).
In the ligation experiments, we observed a clean and fast
ligation reaction as well as strong template and temperature
effects. At 258C and 358C we observed a significant template-
dependent conversion already after a reaction time of 30 s.
After 5 min the templated ligation (1 equiv) is almost
complete. Furthermore, for the nontemplated ligation, no
product formation was observed after 5 min. At 458C, which
Scheme 1. The thio-DNA ligation cycle. A) Overall ligation cycle.
B) Ligation reaction with 3’-activation.
2
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Figure 3. Gel mobility analysis of the templated ligation reaction at
258C (A), 358C (B), 458C (C). Left: Reaction after 30 s. Right: Reaction
after 5 min. Lane 1: nontemplated reaction, lanes 2–5: 0.1 equiv,
0.2 equiv, 0.5 equiv, and 1 equiv of template TACTTCAAGCGGTATAAT,
lane 6: 1 equiv of template TACTTCAAGCTGTATAAT, lane 7: 6-FAM
precursor ON, lane 8: reduced 6-FAM precursor ON. All reactions
were performed in 0.1m phosphate buffer (pH 7) containing 100 mm
NaCl with 0.5 mm precursor concentration. Template concentration
was varied from 0 to 0.5 mm. Ligation reactions were stopped by
addition of a 50-fold excess of Ellman’s reagent in a saturated urea
solution after 30 s and 5 min, and analyzed by denaturing polyacryl-
amide gels by fluorescence detection.
Figure 4. Kinetics of 3’-activated template-directed ligation reaction at
258C (A), 358C (B), 458C (C). The template concentration was varied
from 0 mm to 0.5 mm and the influence of template on the ligation was
investigated for sets of measurements with varying template concen-
trations at 258C, 358C, and 458C. Left: Fluorescence data and
theoretical curves obtained with the FRET detection method making
use of quencher release during ligation. For each set, the rate
constants for the nontemplated (k1) and the templated reaction (k2)
were estimated, as well as the constants for product–template
dissociation (k3) and precursor–template dissociation (k4) using the
SimFit[22] program and Scheme 2. In each series, the measurements
were normalized by calibration for 0% and 100% conversion.
is the melting temperature of the product–template complex
(see the Supporting Information), product formation is
lowered in accordance with the reduced template effect.
Despite the elevated temperature, the nontemplated product
formation is still low after 5 min. The formation of side
products with the fluorescent probe was not observed.
Selectivity for the templated ligation was also evidenced
by ligation experiments with a mutated template (Figure 3A–
C, lanes 6). A change in the sequence of the template by one
base, in close proximity to the ligation site, leads to lower
product formation than that achieved with the matching
template. This effect intensifies with elevated temperature. In
this context several template variations were tested, and all
exhibited a reduced template effect (see the Supporting
Information).
Scheme 2. Reaction model for template-directed ligation; A,B: precur-
sors, C: product, Q: leaving group, T: template, k: fixed rate constants.
To quantify the kinetics, templated ligation was also
monitored directly by fluorescence measurements (Figure 4).
An excess of thiol-ON was used to increase the reproduci-
bility and reduce the effects of auto-oxidation within a series
of measurements, without inducing side reactions. An addi-
tional quenching influence of the template on the product and
the precursor was observed (hybridization quenching) and
quantified by calibration measurements (for details see the
Supporting Information).
The magnitudes of k3 and k4 (Scheme 2) were estimated
based on dissociation constants obtained from melting curves
and the predicted association rate constants k and were
employed in the fit. The melting temperatures for the
precursor–template and product—template complexes were
determined to be 29 and 458C, respectively (see the
Supporting Information), the latter being about 78C lower
than that for the natural phosphodiester sequence. The
background fluorescence as well as the contribution of
hybridization quenching to the fluorescence signal were
taken into account as correction factors in the fit. In this
way, rate constants and product concentrations (Table 2,
Figure 4, right) were directly derived from the observed
fluorescence measurements (for details see the Supporting
Information).
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Table 2: Rate constants and estimated errors for templated ligation
experiments of A and B with 3’-activation as a function of temperature.[a]
Applications to electronically controlled replication systems
and synthetic systems coupling replication and redox metab-
olism are being investigated.[26]
T [8C]
258C
358C
458C
k1 [mÀ1 sÀ1
]
17.5Æ2
(2.7Æ2)ꢂ10À1
(4.7Æ6)ꢂ10À6
5.3Æ3
29.3Æ0.1
1.69Æ0.01
(3.7Æ0.2)ꢂ10À3
39.7Æ0.1
2.2
39.1Æ0.7
6.6Æ0.1
(1.3Æ0.1)ꢂ10À2
820Æ5
k2 [sÀ1
k3 [sÀ1
k4 [sÀ1
]
]
]
Experimental Section
Synthesis of 5’-thiol-ON: Reverse amidites (ChemGene) were
reacted for 5 min on a DNA synthesizer (Gene Assembler Plus)
with 5-benzylmercaptotetrazole (emp Biotec) as activator. Standard
protocols for detritylation, capping, and oxidation were applied.
ON purification: After cleavage from the solid support (conc.
ammonia, 558C, 16 h) products were reduced with a 100-fold excess of
TCEP in buffered solution (100 mm in 0.1m HEPES; pH7) for 15 min.
ONs were purified by RP-HPLC.
r.m.s.[b] [%]
1.9
4.3
[a] A significant temperature variation for both the templated ligation
and the rate constants of dissociation can be deduced. [b] r.m.s. is the
(percentage) ratio root mean square for all species in the overall fit.
The plots and the rate constants reveal a marginal
turnover for the ligation system, a very fast templated ligation
pathway, and a moderate background reaction. Turnover
numbers (TONs) were determined by fluorescence measure-
ments in a series of experiments at various temperatures (see
the Supporting Information). In the presence of 0.01 equiv-
alents of template (5 nm) we observed a TON of up to 6 after
100 min. Other groups have reported TONs of up to 402 over
24 h with the addition of 0.01 mol% template and recently
a TON of 1500 over 15 h was demonstrated with 0.001 mol%
added template.[9c,12]
At 258C we observed a rapid ligation with 50% yield
within the first 60 s and an almost quantitative conversion
after 5 min. The nontemplated ligation accounts for about
5% conversion after 30 min. Due to the rate of the ligation,
the initial rate acceleration can be estimated to be at least 300-
fold.
Synthesis of dabcyl-linker-ON: A 20-fold excess of the dabcyl-
Lys-DTNB linker 13 was added to the reduced thiol-ON (with
a
terminal 6-fluorescein-phosphoramidite modification (Glen
Research)) (0.1m HEPES; pH 7). After 30 min, the product was
isolated by RP-HPLC and desalted with OASIS-HLB cartridges
(Waters). The product was characterized by MALDI-TOF MS with 3-
hydroxypicolinic acid (3-HPA) as matrix.
Ligation reactions: Ligations were performed in 0.1m degassed
phosphate buffer (pH 7) containing 100 mm NaCl. The fluorescence
increase on reaction was monitored using a Varian Cary Eclipse
Spectrometer (excitation 495 nm; emission 515 nm). To start the
reaction, 300 mL of buffered and temperature-annealed ON solutions
were mixed in a 500 mL fluorescence quartz cuvette (path length
10 mm). Ligation reactions for gel analysis were performed with
a reaction volume of 20 mL. To halt the reaction, 10 mL aliquots were
quenched with a 50-fold excess of Ellmanꢀs reagent in 10 mL of
a saturated urea solution. Samples were analyzed by denaturing
PAGE (16%, 120 V, 1.5 h, TBE buffer).
With increasing temperature, up to the melting temper-
ature of 458C, the template effect is weakened: At 508C no
template effect was observable. Since the background reac-
tion is apparently only marginally affected by temperature,
temperature can be used to reduce the overall ligation rate
and to control the templated ligation by influencing hybrid-
ization.
Received: December 8, 2013
Revised: January 28, 2014
Published online: && &&, &&&&
Keywords: chemical ligation · FRET · kinetics ·
.
oligonucleotides · sulfur
We showed that the disulfide-exchange reaction is one of
the fastest ligation reactions, exhibiting half-lives for the
template-directed synthesis on the order of seconds. It is
comparable with enzymatic reaction with ligases, with nucle-
ophilic substitution reactions of thiolated ONs, and with the
DNA-templated native chemical ligation of PNA.[13,23] The
choice of 3’-activation can very effectively reduce background
reactions, whereas in the templated case the proximity and
proper orientation of the reactive sites promotes the ligation
speed. The ligation reaction is also clean—side products were
not observed—and is compatible with elevated temperatures.
Interesting applications could arise from the strong pH
dependency of the disulfide-exchange reaction as well as from
its reversibility. In the context of self-replication[24] and
molecular evolution a fast and externally controllable ligation
chemistry can be highly valuable. The reversibility of the
ligation could be used to create dynamic combinatorial
libraries of disulfides on an ON basis.[25] Significant turnover
could be induced by temperature cycling. There is evidence
for sequence selectivity, especially at elevated temperatures,
which could lead to applications in SNP detection. The
breadth of the ligation method needs to be demonstrated by
sequence variation to natural samples (e.g. Ras gene).
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Angew. Chem. Int. Ed. 2014, 53, 1 – 6
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
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.
Angewandte
Communications
Communications
Chemical DNA Ligation
Thiolated oligonucleotides show unpre-
cedented high reaction rates in chemical
ligation experiments. The influence of
temperature on the templated reaction is
shown with a system designed for fluo-
rescence monitoring. Side reactions and
the nontemplated ligation reactions are
efficiently suppressed by the choice of
DNA modification and the activation site.
V. Patzke,* J. S. McCaskill,
G. von Kiedrowski
&&&& — &&&&
DNA with 3’-5’-Disulfide Links—Rapid
Chemical Ligation through Isosteric
Replacement
6
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
These are not the final page numbers!