Tuning DNA stability to achieve turnover in template for an enzymatic ligation reaction

Article abstract of DOI:10.1002/anie.201102579

Learning to let go: Introducing destabilizing modifications into a DNA template leads to turnover in a DNA-templated ligation reaction. By incorporating a cross-catalytic cycle, self-replication was also achieved, with one target able to make 32 copies of itself (see picture). This destabilization approach represents a general method for incorporating amplification into DNA ligation processes using T4 DNA ligase.

Full text of DOI:10.1002/anie.201102579

Communications
DOI: 10.1002/anie.201102579
Engineering DNA Turnover
Tuning DNA Stability To Achieve Turnover in Template for an
Enzymatic Ligation Reaction**
Abu Kausar, Rosalie D. McKay, Jade Lam, Rohan S. Bhogal, Alexandra Y. Tang, and
Julianne M. Gibbs-Davis*
In the last decade, DNA-based systems have been developed
that are capable of performing sophisticated functions
initiated by molecular recognition. Key examples are the
DNA walkers where directional motion or load pick-up,
transfer, and release are achieved with molecular and spatial
selectivity.[1,2] Attempts to develop autonomous DNA self-
replicating systems, however, without specific sequence
requirements,[3] have in recent years lagged behind.[4–6] As
one of the hallmarks of organisms is their ability to amplify
information and materials through biocatalysis and self-
replication,[6,7] the development of truly biomimetic systems
capable of integrated functions requires incorporating am-
plification into self-assembly and nanotechnology.[8] Not only
do replicating DNA systems provide tools for DNA-based
nanotechnology[8] and insights into the origins of life,[6,7,9] but
Scheme 1. Isothermal turnover in DNA-templated ligation reactions
using destabilizing templates.
they also can be used to isothermally amplify signal in DNA
detection, which can simplify the requirements for point-of-
care diagnostics.[10]
One method for introducing amplification into DNA-
based systems involves generating turnover in DNA-tem-
plated processes.[11] To achieve turnover, the DNA template
that facilitates the reaction of two complementary fragment
strands (Scheme 1, steps A and B) must dissociate from the
product after it has formed (Scheme 1, step C). For ligation
reactions, however, turnover is minimal under isothermal
conditions owing to the enhanced affinity of the template for
the ligated product.[11] Consequently, many detection strat-
egies have focused on template-triggered scission and transfer
reactions rather than ligations to avoid this product inhib-
ition.[11] One method for introducing turnover into ligation
reactions exploits the sensitivity of DNA to destabilizing
modifications present in the middle of a duplex.[5,12,13] By
ligating strands at such a destabilizing site, one can modulate
the stability of the hybridization complex without changing
the temperature or any other reaction condition.[14] If the
stabilities of the complexes before and after ligation are
properly balanced, isothermal turnover should be achieved.
Herein we report a general strategy for achieving turnover
in template for ligations using T4 DNA ligase. Utilizing
modified DNA template strands, the product duplex is
destabilized after ligation. As a result, the template strand is
released, freeing it to template more ligation reactions.
Introducing turnover into simple, enzymatic ligation reactions
provides an avenue for amplifying DNA-based assemblies
constructed by enzymatic ligation.[15–17] Isothermal ligation
strategies also have potential applications in cross-catalytic
replication of DNA, which represents a general method for
amplifying any DNA sequence.[6,7]
Two of the most successful examples of isothermal
turnover in nonenzymatic, chemical ligation systems were
reported by Kool and Seitz, which proved useful in DNA and
mRNA detection.[11,13] In the Kool system, destabilization was
introduced in a ligating fragment strand by adding an alkyl
group between the terminal nucleotide and the electrophilic
end.[18] After ligation with a nucleophilic fragment using
target DNA as a template, the resulting alkyl bridge caused
the product duplex to dissociate. With this example in mind
we synthesized DNA templates containing short alkyl chains
in place of a complementary nucleotide (Figure 1). We also
investigated a model abasic DNA lesion known to destabilize
DNA duplexes (Figure 1, Ab).[19] Previously, PNA analogues
of abasic groups have been demonstrated by Seitz to avoid
product inhibition[20] and improve selectivity[21] in chemical
ligation systems using PNA. Here, the abasic group results in
a template that is missing a base but still contains the
canonical phosphate-sugar backbone.
[*] A. Kausar, R. D. McKay, J. Lam, R. S. Bhogal, A. Y. Tang,
Prof. J. M. Gibbs-Davis
Department of Chemistry, University of Alberta
Edmonton, AB, T6G 2G2 (Canada)
E-mail: gibbsdavis@ualberta.ca
Homepage: http:/www.chem.ualberta.ca/~gibbsdavis
[**] A.K., R.D.M., and J.L. contributed equally to this work. We
acknowledge the Canada Foundation of Innovation and NSERC of
Canada for financial support. We also thank Gareth Lambkin, Prof.
Robert Campbell, and Dr. Brian Stepp for helpful discussions. We
gratefully acknowledge Jing Zheng in the University of Alberta Mass
Spectrometry Facility for performing the MALDI-TOF experiments.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201102579.
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the position of the nicked site and destabilizing group are
given in Figure 2C. To illustrate the extent of destabilization,
we compared these results with the behavior of a perfectly
complementary system (Figure 2A and B, black solid traces,
where D = thymidine). For all of the destabilizing templates,
the decrease in Tm between the nicked and corresponding
product duplexes was 18–228C. In contrast, the Tm difference
was 278C for the natural DNA system (Figure 2D). The
smaller DTm for the destabilized templates suggested that a
temperature might be found where hybridization of the
nicked duplex (Scheme 1, step A) and dissociation of the
product duplex (Scheme 1, step C) were both possible. It still
remained to be seen, however, whether the T4 DNA ligase,
known to ligate mismatched DNA,[23] would tolerate non-
natural modifications on the DNA template strand.
In our ligation experiments, the template was mixed with
the complementary reactive strands (containing a 3’-OH and
5’-phosphate at the ligation site) and 1 unit of T4 DNA ligase
for each 15-mL reaction.[24] We labeled the 5’ end of one of the
fragments with fluorescein to follow ligation by fluorescent
imaging after separation by denaturing polyacrylamide gel
electrophoresis (PAGE). The gel image in Figure 3A illus-
trates the ligation mixtures with one equivalent of template
after 20 h of reaction time, where a product band was evident
for all of the destabilizing templates. This result is significant
since it proves that T4 DNA ligase tolerates nonnatural
modifications to the DNA template near the site of ligation. It
is important to note that ligation with all of the destabilizing
templates was hampered when the ligation site was opposite
the 5’ end rather than the 3’ end of the destabilizing group (see
Supporting Information, Figure S1).
Figure 1. The nicked site prior to ligation. The destabilizing templates
contain a modification (D) in place of a thymidine. The perfect
template contains the complementary thymidine (T).
Achieving the greatest turnover requires that the product
duplex be less stable than the nicked duplex (Scheme 1).
However, because of multivalency[22] the product duplex is
invariably more stable, even with the destabilizing modifica-
tion. As a result, a more reasonable goal is to introduce a
modification that renders the product and nicked duplexes
closer in stability, so that a temperature can be found where
both can form but remain labile. To determine whether the
duplex stabilities of our destabilizing templates were optimal,
we monitored their thermal dissociation behavior. The
temperature at which half the duplex has dissociated is the
melting temperature (Tm), providing a way to compare duplex
stabilities.
To determine whether these destabilizing templates could
turn-over in the reaction, we next monitored the ligation
reaction with substoichiometric amounts of template. At
The thermal dissociation curves are shown for the nicked
duplexes (template + two fragments) and the product
duplexes (template + product) in Figure 2A and B, respec-
tively. The 18-base sequence used in these experiments and
Figure 3. Template labels: – no template; T thymidine; del deletion; Et
ethyl; Bu butyl; cB cis-butenyl; Xy xylyl; Ab abasic. A) Fluorescent
images of denaturing polyacrylamide gels for ligation mixtures using
fluorescein-labeled thymine (TF) with 1 equiv template. B) Turnover
number (TON) versus temperature for ligations with 0.01 equiv
template. C) Percent yield versus time with 1 equiv template. D) TON
versus enzyme concentration (1 unit vs 5 unit) with 0.01 equiv tem-
plate at 248C. Conditions unless otherwise noted: 1 equiv (1.4 mm) TF-
labeled reactant, 1 unit T4 DNA ligase, 20 h, 168C.
Figure 2. The thermal dissociation profiles of A) nicked duplexes
(template:fragments) and B) product duplexes (template:product).
[DNA]=1.3 mm per strand (pH 7.0, 20 mm PBS, 10 mm MgCl2).
C) Sequence of strands where D is thymidine or a destabilizing group.
D) Table of dissociation (melting) temperatures (Tm).
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208C, when 0.01 equivalents of complementary template (T)
the Seitz group using similar probe (1.2 mm) and template
(12 nm) concentrations.[20] Although both Kool[18] and Seitz[20]
report higher TONs (91.6 and 226, respectively) these were
only achieved by increasing the fragment:template ratio to
10000, which facilitates dissociation of the product duplex.
This required that they use HPLC or radio-imaging with
PAGE, which allowed them to reliably distinguish 0.0916%
or 0.226% of product forming from reactions with non-
negligible background. In contrast, our approach has the
advantage of no background reaction when template is
absent. Moreover, T4 DNA ligase, standard DNA ligating
groups, and the commercially available abasic destabilizing
modifications should prove more accessible to nonsynthetic
labs than these chemical methods.
This methodology for using catalytic amounts of template
to amplify a DNA-ligated material made by T4 DNA ligase
can be applied in smart, DNA-based systems. For example, an
environmental stimulus can be used to release destabilizing
template causing the amplification of a DNA material, like
DNA-ligated gold nanoparticle aggregates.[16] If, however,
one wanted to use this principle of destabilization for native
DNA detection, another complementary ligation cycle must
be included. Scheme 2 illustrates a cross-catalytic replication
strategy that can be initiated by a native DNA strand
representing a target sequence. In this set of experiments,
the destabilizing template is formed in situ by a ligation
reaction templated by the natural DNA target (Scheme 2,
steps A and B). As a result of ligation, the same product
duplex is formed as in the previous cycle. Consequently, the
product:template duplex should be destabilized leading to the
release of the original target and the newly formed destabi-
lizing template (Scheme 2, step C). This destabilizing tem-
plate can now generate a copy of the original target template
(Scheme 2, steps D and E), which goes on to catalyze the
formation of more destabilizing templates. As the product of
were used only 1.3% of the fluorescent fragment strand was
ligated, indicating that the dissociation of the perfect product
duplex was unfavorable. In contrast, using the same amount
of an abasic template (Ab) led to 3.2% of the ligated product.
From the ratio of [product]/[template] the turnover number
(TON) was calculated. As shown in Figure 3B, the degree of
turnover for the perfect template (T) was between 1 and 2 for
all reaction temperatures using 0.01 equivalents of template.
In contrast, the turnover was greater than 2 for several of the
destabilizing templates. The only inactive templates that
yielded little or no ligated product under these substoichio-
metric conditions were the xylyl template (Xy) and a template
containing a deletion of the thymidine (del, data not shown).
With the cis-butenyl template, turnovers were between 0.9
and 1.7, which indicated that this rigid linkage did not
promote catalytic behavior.
For all of the active destabilizing templates, the highest
TON was observed at 288C, well above the melting temper-
ature of their corresponding nicked duplexes (Figure 3B).
Specifically, the highest TON observed at this enzyme
concentration was 5.0 for the abasic template (Figure 3B,
Ab). To explain the temperature trends, we propose that the
decrease in nicked duplex stability at 288C is compensated for
by the higher rate of ligation or dissociation of the product
duplex. At temperatures higher than 288C, however, the
decrease in TON suggests that the formation of the nicked
duplex becomes unfavorable. Importantly, at all temper-
atures, no background ligation was observed in the absence of
template (Figure 3A, –). The enzyme requirement that the
DNA be double-stranded eliminated any background reac-
tion illustrating a major advantage to this approach. More-
over, these results also suggest that employing a catalyst
which favors double-stranded DNA might be a way to avoid
non-templated background in chemical ligations.[5]
To see how the destabilizing template influenced the rate
of ligation, we measured the yield versus time in ligations
using one equivalent of template. As shown in Figure 3C,
within 10 min ligation is complete when natural DNA
template (T) is used.[25] Most of the destabilizing templates
are only a little slower with the Ab and cB template requiring
20 min, and the other templates requiring less than 40 min.[26]
Comparing our results with those of chemical ligation
methods that demonstrate lower rates of ligation[18,27] indi-
cates that faster ligation methods lead to greater turnover,
which is consistent with previous reports on isothermal
chemical ligation methods.[20,21]
In the experiments described above we used the typical
ligase concentration for ligating nicked duplexes of 1 unit
enzyme per equivalent of fluorescent fragment strand (1.4 mm,
15 mL). To see whether increasing enzyme concentration
would increase the amount of turnover, ligation reactions
using concentrated enzyme (5 units per reaction) were
performed with the same amount of template (0.01 equiv-
alents). At higher enzyme concentration, the perfect DNA
template (T) still exhibited a TON close to one. In contrast,
the Bu and Ab templates generated 18 product strands per
template (Figure 3D), which is 5-fold higher than the
maximum turnover number of 3.5 previously reported by
Scheme 2. Cross-catalytic cycles with destabilizing fragments.
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each cycle is a template for the other, significant amplification
of the original target should ensue.
adenine thus minimizing nonspecific joining of the fragments.
Consequently, very little background ligation is observed.
In conclusion, we have demonstrated that DNA templates
for enzymatic ligation reactions can turn-over in the ligation
cycle by introducing a destabilizing modification into the
template strand. This work complements previous studies
illustrating how destabilizing fragments can be used to
facilitate turnover.[11,13] Additionally, the turnover numbers
observed in our system are higher than the best isothermal
chemical ligation strategies at similar strand lengths, concen-
trations, and ratios.[11,13] We have also taken this idea a step
further by adding in another cycle that leads to a self-
replicating DNA system using destabilization to overcome
product inhibition. The success of the Ab templates in both
cycles is especially promising as this phosphoramidite is
commercially available allowing simple access to templates
capable of turnover. Not only will this strategy enable
advances in nanotechnology in the replication of DNA
materials, but it should also be useful in isothermal target
amplification for the broad field of DNA diagnostics. Finally,
with simple destabilizing groups and rapid ligation methods,
turnover and target-initiated self-replication are now possi-
ble, which should reinvigorate interest in autonomous DNA
replication.[4–6] We are currently working on expanding this
destabilization approach to other rapid ligation systems using
nonenzymatic strategies.
To verify that we could observe cross-catalysis, we
combined the fragment strands listed in Scheme 2, including
a fluorescent modified fragment corresponding to the bottom
cycle (TF, fluorescein-modified thymine). We then introduced
a target DNA sequence which, although active in the top
cycle, should have no effect on the fluorescein-labeled
fragment. Therefore, the formation of any fluorescent ligated
product would signify cross-catalysis. Previous reports sug-
gested that T4 DNA ligase would ligate a destabilizing
fragment terminated with a 5’-phosphate abasic group.[28]
Indeed, as shown in Figure 4 target-initiated cross catalysis
Figure 4. SC: Single-catalyst cycle using an abasic destabilizing tem-
plate and fragments. CC: Cross-catalytic cycle using target as template
and fragments, one of which contained an abasic group. Conditions:
0.01 equiv template (14 nm); *0.001 equiv (1.4 nm); 1 unit T4 DNA
ligase; 248C; 20 h.
Experimental Section
Preparation of DNA strands: DNA was synthesized on an ABI 392
solid-phase synthesizer using Glen Research reagents. Strands were
purified by Glen-Pak DNA Purification cartridges (cat. 60-5200-01)
according to the DMT-On protocol. Standard nucleotide phosphor-
amidite and the following were used: Chemical Phosphorylation
Reagent II (cat. 10-1901-90), Fluorescein-dT Phosphoramidite (cat.
10-1056-95), and dSpacer CE Phosphoramidite (cat 10-1914-90) for
the abasic (Ab) template and fragments. All other destabilizing
templates were prepared from the corresponding protected diols with
solid-phase synthesis. OligoCalc (http://www.basic.northwestern.edu/
biotools/oligocalc.html) was used to determine the extinction coef-
ficients where the destabilizing templatesꢀ absorptivity was assumed
to be Ac, with D = a deletion.
occurred for the Ab-substituted system. Not only did we
observe self-replication in this two-cycle system, but the
exhibited cross-catalytic TON (defined as [product]/[initial
template]) was greater than that for the single catalytic cycle
(TON 14 vs 4.5; Figure 4, CC vs SC, respectively). Decreasing
the number of equivalents of template (CC*) further
increased the number of cross-catalytic turnovers, indicating
that dissociation is favored as the fragment:template ratio
becomes larger.[18,20] The highest cross-catalytic turnover of 32
corresponds to the target undergoing on average 32 cycles of
self-replication (Figure 4, CC*). As a point of reference, our
system exhibits the same number of self-replication cycles as
shown by Albagli et al. in one of the few reported cross-
catalytic chemical ligation systems applicable to DNA
amplification and detection.[29] In their system, however,
temperature cycling was used to achieve turnover, whereas
our system requires no mechanical or thermal intervention.
For the cross-catalytic reactions using 1 unit of ligase and
the 5’-phosphate abasic fragment, we observed a small level of
background (0.5% and 3.7% product at 20 and 248C,
respectively, with no template present).[30] In contrast, the
5’-phosphate thymidine fragment exhibited a large amount of
background (25% ligation product at 208C) (see Supporting
Information, Figure S2). The reason for the high background
in the natural system is based on our probe design, which
leads to a one-base overhang of thymidine on one fragment
and deoxyadenosine on the other. Although this overhang
exists in our Ab-modified system, replacing the thymidine
with the abasic group prevents hydrogen bonding with the
Strands:
Ac
3’-AACAATTTA-D-AACTATTC-5’; D = T
or destabilizing group
A
C
3’-GAATAGTT-A-TAAATTGTT-5’
3’-GAATAGTTA-5’
CP
B
3’-GAATAGTTAPhosphate-5’
3’-TAAATTGTT-5’
BF
3’-TAAATTGTTFluorescein-5’
3’-AACTATTC-5’
3’-AACAATTTATPhosphate-5’
3’-AACAATTTA(Ab)Phosphate-5’
Cc
BcP
BcAbP
Thermal dissociation experiments: 1.3 nmol of each DNA
sequence (Ac and A for the product duplex experiments and Ac, B,
and C for the nicked duplex experiments) were combined in PBS
buffer (1.0 mL, 10 mm MgCl2, 20 mm pBS, pH 7.0) and hybridized for
ca. 15 min. While stirring at 100 rpm, absorbance readings at 260 nm
were taken from 10 to 608C at 18C intervals, with 1 min hold time.
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Ligation experiments. Strand amounts: Single-cycle reactions: BF
[10] A. R. Connolly, M. Trau, Angew. Chem. 2010, 122, 2780 – 2783;
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1 equiv; CP 2 equiv; template (Ac) 1 or 0.01 equiv. Cross-cycle
reactions: BF 1 equiv; CP 2 equiv; A 0.01 or 0.001 equiv; Cc 2 equiv;
and 2 equiv of either strand BcP or BcAbP. In a typical ligation, where
1 equiv = 21 pmol, the appropriate amounts of DNA fragments and
template were first combined in water in a 400 mL minicentrifuge tube
to reach a final volume of 10 mL and incubated at the desired reaction
temperature. While several of these DNA solutions incubated, in a
separate mini-centrifuge tube, T4 DNA ligase (8 mL) at lower
concentration (1 unitmLÀ1, Invitrogen cat. 15224-017) or higher
concentration (5 unitmLÀ1, Invitrogen cat. 46300-018) was mixed
with ligation buffer (24 mL, 5 ꢁ concentrated) and water (8 mL). A
portion of this ligase mixture (5 mL) was immediately added to each of
the DNA solutions (final [DNA] = 1.4 mm for each equivalent). The
reactions were then placed in a covered thermal incubator for 20 h
unless otherwise noted. To stop ligation, EDTA(aq) (1 mL, 0.5m) was
added for every unit of enzyme present. For the kinetic experiments,
aliquots (3 mL) were removed from the bulk ligation mixture at
various reaction times and placed in a separate microcentrifuge tube
containing EDTA(aq) (1 mL, 0.5m). Samples were stored at 48C until
analyzed by 15% denaturing PAGE.
[11] T. N. Grossmann, A. Strohbach, O. Seitz, ChemBioChem 2008, 9,
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[15] M. Mendel-Hartvig, A. Kumar, U. Landegren, Nucleic Acids
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[16] S. A. Claridge, A. J. Mastroianni, Y. B. Au, H. W. Liang, C. M.
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Received: April 14, 2011
Published online: August 17, 2011
[24] One unit is the amount of enzyme needed to catalyze the
conversion of 1 nmol of 32P-labeled pyrophosphate into ATP in
20 min at 378C (Invitrogen).
Keywords: DNA-templated ligation · engineering turnover ·
isothermal DNA amplification · self assembly · self replication
.
[25] The rate of ligation for perfect template should first be measured
to verify the enzyme activity. If the enzyme is less active due to
improper storage or handeling, no turnover is observed.
[26] We attribute the leveling off of the signal for the ethyl and butyl
templates to subtle differences in the extinction coefficients used
to calculate the DNA concentration. The leveling off of the xylyl
suggests that most of the xylyl template is in a conformation that
does not allow for hybridization or ligation.
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[29] D. Albagli, R. V. Atta, P. Cheng, B. Huan, M. L. Wood, J. Am.
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