Small molecule induced control in duplex and triplex DNA-directed chemical reactionst

Article abstract of DOI:10.1039/b919387a

Triplex DNA binders can effectively control copper-catalysed alkyne-azide click reactions in DNA architecture, such that either duplex or triplex DNA directed reactions of terminally attached azides and alkynes occur, in the absence or presence of triplex

Full text of DOI:10.1039/b919387a

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/obc | Organic & Biomolecular Chemistry
Small molecule induced control in duplex and triplex DNA-directed chemical
reactions†
Mikkel F. Jacobsen, Jens B. Ravnsbæk and Kurt V. Gothelf*
Received 17th September 2009, Accepted 27th October 2009
First published as an Advance Article on the web 9th November 2009
DOI: 10.1039/b919387a
Triplex DNA binders can effectively control copper-catalysed
alkyne–azide click reactions in DNA architecture, such that
either duplex or triplex DNA directed reactions of terminally
attached azides and alkynes occur, in the absence or presence
of triplex DNA binder, respectively.
quantitatively,12,13 and notably DNA degradation was avoided by
including a ligand for copper.
To demonstrate the small-molecule-induced control of the
CuAAC reaction we have applied a dialkyne–DNA conjugate,
an azide–DNA conjugate and a triplex forming oligonucleotide
(TFO) azide conjugate (Scheme 1). The depicted triplex DNA is
unstable at room temperature in the absence of a triplex DNA
binder and duplex control prevails leading to only one click
reaction.
In the presence of a triplex DNA binder triplex control takes
over forming the parallel pyrimidine–purine–pyrimidine triplex
with Hoogsteen base pairing to the triplex-forming oligonu-
cleotide (TFO), leading to two click reactions. Thereby, the central
dialkyne-modified oligonucleotide becomes ligated to the two
azide-modified oligonucleotides in one step via 1,2,3-triazole head
groups formed in the double click reaction resulting in non-
symmetrical three-way branched oligonucleotides.
Nature utilizes a multitude of macromolecular recognition ele-
ments to control chemical reactivity such as nucleic acids and
proteins.1 For example, it is well-known that the reactivity of
allosteric enzymes can be modulated by the recognition of small
molecule allosteric regulators through changes in conformation.2
A plethora of different small molecules bind to various nucleic
acid architectures inducing stabilization.3 This includes higher
order structures such as G-quadruplex and triplex DNA. Along
these lines, triplex DNA binders are small and usually cationic
molecules which stabilize triplex DNA and often possess highly
selective binding to triplex over duplex DNA.4,5
A selection of azide and dialkyne-modified oligonucleotides was
synthesized (Fig. 1, Supporting Information). Both azide (X1,
X2) and dialkyne modifications (conjugated and non-conjugated
dialkyne, Y1, Y2 and Z1, Z2, respectively) were investigated with
two different lengths of linkers (C6 or C6+C12) (O1–O8). Since
the degradation of DNA in the presence of Cu(I) and oxygen can
seriously affect the outcome of any CuAAC reaction,14 ligands for
Cu(I) were employed to protect it from oxidation and partly shield
the DNA. Two different ligands, BPDS (1)15 and THTA (2)16 were
tested. For binding and stabilization of triplex DNA two triplex
It has been demonstrated by Mao et al. how DNA-triplex
structures containing CGC triplets can be used in a pH regulated
triplex formation to control the formation of an amide bond.6
Here we have used triplex DNA as a model system to demonstrate
how the non-covalent interaction of a small molecule regulator,
in this case a triplex DNA binder, can be used to control the
covalent chemical reactions of conjugated functional groups. The
recent surge in the use of DNA-directed chemistry,7 e.g. in drug
discovery,8 may benefit from the development of reactions based
on novel and controllable higher-order DNA architectures.
The Huisgen–Sharpless–Meldal copper-catalyzed 1,3-dipolar
cycloaddition (CuAAC) has been used extensively for the
modification of nucleosides (e.g. conjugation of an fluo-
rophor/biomolecule) prior to or following their incorporation
into oligonucleotides.9,10 Contrarily, only very few examples of
DNA-directed CuAAC reactions have been reported, in which an
alkyne–DNA conjugate and an azide–DNA conjugate, in low
concentrations are brought in close proximity either by direct
hybridization or by hybridization of the two conjugates to a
template strand.7 In two papers Liu et al. performed DNA-
directed CuAAC reactions, and in both cases moderate yields
were obtained in part due to DNA degradation in the presence of
Cu(I).11 More recently Brown et al. described a chemical ligation
type DNA-directed CuAAC reaction which proceeded almost
Danish Research Foundation: Centre for DNA Nanotechnology at the
Interdisciplinary Nanoscience Center and the Department of Chemistry,
AarhusUniversity, Langelandsgade 140, 8000 Aarhus C, Denmark. E-mail:
kvg@chem.au.dk; Fax: 86196199
† Electronic supplementary information (ESI) available: Experimental
procedures, MALDI-TOF and RP-HPLC data, and spectral data for all
new compounds. See DOI: 10.1039/b919387a
Fig. 1 Oligonucleotides, ligands, and triplex DNA binders prepared
and/or applied in this study.
50 | Org. Biomol. Chem., 2010, 8, 50–52
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
Scheme 1 Controlling nucleic acid-directed chemical reactions with triplex DNA binders.
DNA binders, coralyne chloride (3)4c and the more potent triplex
DNA binder 44d were employed.
which corresponds to the ligation of the TFO strand and one of
the duplex strands (Hoogsteen product, lane 4). Thus, the strength
of stabilization with the triplex DNA binders is reflected in the
reaction outcome.
Triplex formation is a kinetically slow process compared to
duplex formation. Therefore, preformed duplexes were subjected
◦
to annealing for an extended period (overnight) at 5 C with the
High reaction yields for the formation of the triplex products
from the three strands were obtained in the reaction of both
non-conjugated and conjugated dialkyne-ODNs by employing the
ligand 2 and triplex DNA binder 4 (lane 1 and 2) as indicated by
denaturing PAGE and RP-HPLC. Yields of 90% and 80% for
the duplex (lane 3) and triplex directed reaction (lane 1), respec-
tively, were obtained as estimated by image analysis (GeneTools
software) and corroborated by RP-HPLC analysis.16,17 In the case
of dialkyne-oligonucleotide O6 weak higher bands besides the
triplex product were observed which we assign to oligomerization
reactions (homocouplings) of the dialkyne catalyzed by Cu(I)
(lane 2). Similar faint bands were not observed with the non-
conjugated dialkyne-oligonucleotides O3 and O7.18
TFO strand (and any triplex DNA binder), and subsequently
warmed slowly to 22 ◦C before adding the copper-catalyst mixture
and allowing the conjugates to react for 2 h. Longer reaction times
were not beneficial for the yield of the reactions, due to degradation
of DNA.
The triplex and duplex-directed reactions were analyzed by
denaturing PAGE (Fig. 2). Most prominent is the complete
change in chemical reactivity in the presence (lane 1 and 2)
and absence (lane 3) of triplex DNA binder 4, and the high
selectivity of the reactions. On the other hand, the weaker triplex
DNA binder coralyne (3) led to a less clean reaction with the
contaminant formation of both duplex product and the product
As controls the exclusive formation of duplex product from O5
and O7 was carried out (lane 5), as was the ligation of the TFO
strand O8 with O7 by employing the non-azide-modified duplex
strand O9 (lane 6). As expected the absence of copper catalyst
resulted in no reaction (lane 11).
Optimization of the reaction conditions revealed that subtle
differences in the efficiency of C6 linkers compared to C6+C12
linkers existed, which demonstrates the distance dependence of
the reaction.19 Minor amounts of both Hoogsteen and duplex
product were seen in the case of short linkers (Fig. 2, lane 7), while
the combination of short linkers on the duplex strands and a long
linker on the TFO strand also gave a clean reaction (lane 8). The
use of BPDS ligand (1) led to considerably reduced yields due
to degradation of DNA (lane 9), as indicated by the faint band
and confirmed by RP-HPLC, while the absence of ligand barely
provided any product (lane 10).
Fig. 2 Analysis of the CuAAC reactions by denaturing PAGE (reaction
conditions, unless otherwise noted: 1 : 1 : 1 strand ratio; triplex DNA
binder, ligand, CuSO4 and sodium ascorbate in PBS buffer. T1–T3:
intramolecular triplexes).
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 50–52 | 51
©

View Article Online
Table 1 Thermal stability of inter and intramolecular triplexesa
Triplex DNA binder Tm3 → 2/◦C Tm2 → 1/◦C
may be expanded considerably in scope, which is desirable in
other complex DNA-based reaction systems where precise control
of reactivity is required. Furthermore, the method provides a
new high-yielding method for the formation three-way branched
nonsymmetrical DNA sequences that each in principle may be
extended in a unique sequence, and could be useful in the
construction of DNA nanostructures.
Entry Triplex
1
2
3
4
O5+O7+O8
3
29.6
54.0
11.0
53.4
45.6
O5+O7+O8
O5+O7+O8
T1a
4
—
—
41.3
64.0
a Identical values were obtained for T2, T3 and T4 triplexes (T4 is formed
from O1+O2+O4).
Acknowledgements
Financial support for this work by the Danish National Research
Foundation, and the Carlsberg Foundation is gratefully acknowl-
edged.
The identity of the formed intramolecular triplexes was con-
firmed by MALDI-TOF MS, and they could readily be iso-
lated by RP-HPLC, and characterized by thermal denaturation
experiments. The intramolecular triplexes T1–T4 exhibit similar
melting behavior characterized by a weak Hoogsteen transition at
53 ◦C, much higher than their intermolecular counterparts (in the
abs◦ence of triplex binder), and a duplex/random coil transition at
64 C (Fig. 3 and Table 1). Notably, no hysteresis was observed
due to fast hybridization kinetics.
References
1 (a) Nucleic Acids in Chemistry and Biology, 3rd Ed. (Eds: G. M. Black-
burn, M. J. Gait, D. Loakes, D. M. Williams), Royal Society of
Chemistry, Cambridge, 2006; (b) Principles of Nucleic Acid Structure,
S. Neidle, Elsevier, 2008; (c) Proteins: Structure and Function, D. Whit-
ford, Wiley, Chichester, 2005.
2 Allosteric Regulatory Enzymes, T. Traut, Springer, New York, 2008.
3 M. J. Hannon, Chem. Soc. Rev., 2007, 36, 280–295.
4 (a) J. B. Chaires, J. Ren, M. Henary, O. Zegrocka, G. R. Bishop and L.
Strekowski, J. Am. Chem. Soc., 2003, 125, 7272–7283; (b) C. Escude´,
C. H. Nguyen, S. Kukreti, Y. Janin, J.-S. Sun, E. Bisagni, T. Garestier
and C. He´le`ne, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 3591–3596;
(c) J. S. Lee, L. J. P. Latimer and K. J. Hampel, Biochemistry, 1993,
32, 5591–5597; (d) W. D. Wilson, F. A. Tanious, S. Mizan, S. Yao,
A. S. Kiselyov, G. Zon and L. Strekowski, Biochemistry, 1993, 32,
10614–10621; (e) J. L. Mergny, G. Duval-Valentin, C. H. Nguyen, L.
Perrouault, B. Faucon, M. Rouge´e, T. Montenay-Garestier, E. Bisagni
and C. He´le`ne, Science, 1992, 256, 1681–1684.
5 B. P. Belotserkovskii, E. De Silva, S. Tornaletti, G. Wang, K. M. Vasquez
and P. C. Hanawalt, J. Biol. Chem., 2007, 282, 32433–32441.
6 Y. Chen and C. Mao, J. Am. Chem. Soc., 2004, 126, 13240.
7 D. R. Liu and X. Li, Angew. Chem., Int. Ed., 2004, 43, 4848–4870.
8 (a) B. N. Tse, T. M. Snyder, Y. Shen and D. R. Liu, J. Am. Chem. Soc.,
2008, 130, 15611–15626; (b) E. M. Driggers, S. P. Hale, J. Lee and N. K.
Terrett, Nat. Rev. Drug Discovery, 2008, 7, 608–624; (c) L. A. Haff,
C. G. M. Wilson, Y. Huang, B. K. Benton, J. F. Bond, A. M. Stern,
R. F. Begley and J. M. Coull, Clin. Chem., 2006, 52, 2147–2148.
9 J. E. Moses and A. D. Moorhouse, Chem. Soc. Rev., 2007, 36, 1249–
1262.
10 (a) F. Amblard, J. H. Cho and R. F. Schinazi, Chem. Rev., 2009, 109,
4207–4220; (b) P. M. E. Gramlich, C. T. Wirges, A. Manetto and T.
Carell, Angew. Chem., Int. Ed., 2008, 47, 8350–8358.
11 (a) Z. J. Gartner, R. Grubina, C. T. Calderone and D. R. Liu, Angew.
Chem., Int. Ed., 2003, 42, 1370–1375; (b) M. W. Kanan, M. M.
Rozenman, K. Sakurai, T. M. Snyder and D. R. Liu, Nature, 2004,
431, 545–549.
12 R. Kumar, A. El-Sagheer, J. Tumpane, P. Lincoln, L. M. Wilhelmsson
and T. Brown, J. Am. Chem. Soc., 2007, 129, 6859–6864.
13 A. H. El-Sagheer and T. Brown, J. Am. Chem. Soc., 2009, 131, 3958–
3964.
14 (a) R. L. Weller and S. R. Rajski, Org. Lett., 2005, 7, 2141–2144; (b) C. J.
Burrows and J. G. Muller, Chem. Rev., 1998, 98, 1109–1152.
15 W. G. Lewis, F. G. Magallon, V. V. Fokin and M. G. Finn, J. Am. Chem.
Soc., 2004, 126, 9152–9153.
16 T. R. Chan, R. Hilgraf, K. B. Sharpless and V. V. Fokin, Org. Lett.,
2004, 6, 2853–2855.
17 RP-HPLC analysis indicated that the observed non-quantitative con-
version is due to partial degradation of the oligonucleotides.
18 The higher propensity for oligomerisation of oligonucleotides carrying
Y1, Y2 groups compared to Z1, Z2 may be explained by the higher
acidity of the terminal alkynes.
Fig. 3 Thermal denaturation profiles of inter and intramolecular triplexes
at 260 nm (different absorbance scale for T1 and O5+O7+O8 triplexes).
These results are supported by the thermal denaturation profiles
of the triplex O5+O7+O8 (Fig. 3 and Table 1). Hence, the
3 → 2
Tm
value for the weak Hoogsteen transition (triplex/duplex
transition) in the absence of triplex binder is 11.0 ◦C, well below
room temperature, and only the duplex-directed reaction occurs.
On the other hand, in the presence of 4 it raises to 54.0 ◦C,
where stabilization is so pronounced that the Hoogsteen and
duplex/random coil transitions coincide,4d the triplex-directed
reaction is highly favored. Coralyne (3) provides less stabilization
(29.6 ◦C) with a broad Hoogsteen transition closer to room
temperature, and a less selective reaction ensues. Considerable
hysteresis of the melting curves was observed due to the slow
kinetics involved in the formation of intermolecular triplexes.4e
In summary, we have demonstrated the use of triplex DNA
binders as external stimuli for controlling chemical reactivity in
higher-order DNA-directed reactions. Thus, the efficient double
click reactions reported here could be controlled by simply adding
a strong triplex DNA binder such as naphthylquinoline (4), while
selectivity decreased with the weaker binder coralyne (3). Given
the huge number of small molecules available for binding and
stabilization of almost any DNA architecture, the methodology
19 (a) Z. J. Gartner, M. W. Kanan and D. R. Liu, Angew. Chem., Int. Ed.,
2002, 41, 1796; (b) Z. J. Gartner and D. R. Liu, J. Am. Chem. Soc.,
2001, 123, 6961.
52 | Org. Biomol. Chem., 2010, 8, 50–52
This journal is
The Royal Society of Chemistry 2010
©