Fast thiol-maleamic methyl ester addition for facile covalent cross-linking of oligonucleotides

Article abstract of DOI:10.1016/j.tetlet.2013.01.112

Thiol-Michael-cross-linked oligodeoxynucleotides were synthesized through the thiol-maleamic acid methyl ester addition reaction. As a metal-free 'click' reaction, the cross-linked method was fast responsive and highly sensitive. The newly synthesized oligodeoxynucleotides were thermally stable and their global structures retained those of non-cross-linked oligodeoxynucleotides and showed responsive reaction to different DNA restriction enzymes.

Full text of DOI:10.1016/j.tetlet.2013.01.112

Tetrahedron Letters 54 (2013) 1916–1920
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Fast thiol–maleamic methyl ester addition for facile covalent cross-linking
of oligonucleotides
⇑
Liqiang Cao, Zhen Xi
State Key Laboratory of Elemento-Organic Chemistry and Department of Chemical Biology, Nankai University, Tianjin 300071, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Thiol-Michael-cross-linked oligodeoxynucleotides were synthesized through the thiol–maleamic acid
methyl ester addition reaction. As a metal-free ‘click’ reaction, the cross-linked method was fast respon-
sive and highly sensitive. The newly synthesized oligodeoxynucleotides were thermally stable and their
global structures retained those of non-cross-linked oligodeoxynucleotides and showed responsive reac-
tion to different DNA restriction enzymes.
Received 22 October 2012
Revised 3 January 2013
Accepted 18 January 2013
Available online 8 February 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Thiol-Michael-cross-linked
oligodeoxynucleotides
Metal-free ‘click’ reaction
Thermal stability
DNA restriction enzyme
Synthetic oligonucleotides (ODN) are one of the most important
molecular tools for genomic research and biotechnology.1 Recently
much effort has been dedicated to the application of the relatively
short double-stranded oligodeoxynucleotides (ODN) in developing
therapeutic agents as cis-elements, referred to as decoy ODN, to
trap trans-activators, and transcription factors.2 Many bioconjuga-
tion methods for ODN modifications were reported in the litera-
ture.3 And the ‘click’ reaction concept has become important in
efficient ODN modification. These reactions, using two comple-
mentary strands to provide a quantitative yielding reaction, should
ideally be able to be applied under a wide range of mild conditions
and be tolerant to many other functional groups.4 The most com-
monly studied reaction of this family is Huisgen 1,3-dipolar
cyclo-addition of azide and terminal alkyne functionalities, usually
catalyzed by Cu(I) that may show potential nucleic acid damage.5,6
Other reported approaches include Diels–Alder reaction7 or the
oxidation of thiols to form a disulfide.8
The thiol-Michael addition, as a kind of metal-free ‘click’ reac-
tion with fast reaction to form a stable thioether bond and mild
aqueous reaction condition, should be useful for ODN modifica-
tion.9,10 Dumbbell ODN, a circular ODN consisting of a double-
stranded stem region and nucleotide loops at both of their termini,
possessed increased exonuclease resistance.11,12 We have recently
developed a very fast thiol-reactive probe based on thiol-Michael
addition.10 This fast reaction may be beneficial for nucleic acid
modification. In this Letter, the thiol–maleamic acid methyl ester
addition was successfully applied to cross-linking oligodeoxynu-
cleotides. And the capped ODN still showed an excellent response
to different DNA restriction enzymes.
The artificial dumbbell ODN was constructed by using thiol-
containing oligodeoxynucleotides making Michael addition to the
dimmer b (Scheme 1). Dimmer b can be easily synthesized from
available starting materials (see Supplementary data) and is stable
both in the solid state and in DMSO or methanol solution for sev-
eral months. The b-mercaptoethanol addition results supported
that dimmer b reacts with thiols immediately (see Supplementary
data), and the product is stable. The thiol–maleamic acid methyl
ester reaction showed an excellent cross-linking function in the
modifications of ODN, which was employed as a strategy to syn-
thesize decoy ODN (Fig. 1a). The fast reaction between the thiol
O
O
O
O
O
OH
HO
HN
NH
H2N
NH2
CH2Cl2 reflux
O
O
a
O
O
O
HN
O
NH
TsOH.H2O
CH3OH reflux
O
O
dimmer b
⇑
Corresponding author. Tel./fax: +86 22 23504782.
E-mail address: zhenxi@nankai.edu.cn (Z. Xi).
Scheme 1.
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.01.112

L. Cao, Z. Xi / Tetrahedron Letters 54 (2013) 1916–1920
1917
Figure 1. Formation of the thiol–maleamic acid methyl ester cross-linked dsODN by the metal-free ‘click’ reaction: (a) cross-linking of hairpin ODN (hpODN) at a single site
and cross-linking of dumbbell ODN (dbODN) at both termini. (b) Sequences of the ODN used in this study. S: Thiol-modified C6 S–S. NF-
with a blue box, and EcoRI recognition regions are marked with a yellow box.
jB recognition regions are marked
and maleamic acid methyl ester permits the formation of an inter-
molecular bridge.
purification. The MALDI-TOF MS of the PAGE purified cross-linking
gel band showed a single major peak at 16,726 Da, which is consis-
tent with the hpODN formula weight, 16,709 Da [M+Na]+.
The designed double-stranded ODNs are shown in Figure 1b.
The left shaded part is the sequence of NF-
NF- B upregulates genes related to inflammatory and immunolog-
ical responses, such as TNF- , adhesion molecules, macrophage
j
B.13 Activation of
Encouraged by the above results, the thiol–maleamic acid
methyl ester conjugation was used to construct dumbbell ODNs.
ssODN labeled by thiol-modifier C6 S–S group at both the 50-end
and the 30-end (E) was synthesized (Scheme 2). Synthesis of
cross-linked dsODN2 was conducted by the identical procedure
of dsODN1 (Fig. 1). To avoid the intermolecular polymerization,
an optimized concentration that the Michael addition reaction of
thiol–maleamic acid methyl ester conducted only after annealing
j
a
colony-stimulating factor (MCSF), granulocyte/macrophage
colony-stimulating factor (GMCSF), and monocyte chemoattrac-
tant protein (MCP). Therefore, transfection of NF-jB decoy ODN
leads to anti-inflammatory and immuno-suppressive activities.14
The right shaded part is the target sequence of EcoRI. EcoRI is an
endonuclease enzyme isolated from strains of Escherichia coli, as
a part of the restriction modification system15 to check if the termi-
nal base modifications distort the helical geometry of the duplex.
The cross-linking process for dsODN was shown in Scheme 2.
Thiol-modified C6 S–S was first introduced at one side of the
ssODN on DNA synthesizer using a standard phosphoramidite
elongation cycle for coupling of commercial nucleoside phospho-
ramidites.16 Then the thiol protecting group of oligonucleotide A
was removed by tris(2-carboxy-ethyl)phosphine (TCEP) to afford
the oligonucleotide B, which was subsequently treated with satu-
rated solution of dimmer b in methanol at 37 °C for 5 h and puri-
fied by OMEGA MICROSEP 3K to give the oligonucleotide C
(Scheme 2). The cross-linked hpODN was obtained by annealing
was found to be below 1
lM. Thus the equal equivalent of oligonu-
cleotides G and H (Scheme 2) (0.5
l
M) was annealed in 0.1 M
MOPS buffer (pH 8.0) containing 1 M NaCl. Then the dsODN solu-
tion was incubated at 37 °C for 2 h, and monitored by denaturing
PAGE (Fig. 2a). When we incubated the dsODN (0.5 lM) for some
time before annealing, there was no apparent cross-linked product
without annealing (Lane 11 in Fig. 2a). These results implied that
the thiol-Michael addition was carried out during the annealing
process. The MALDI-TOF MS of the PAGE purified cross-linking
gel band showed a single major signal at 17,442 Da, which is con-
sistent with the calculated dbODN formula weight, 17,418 Da
[M+Na]+.
CD spectroscopy was used to study the helical geometry of the
chemically modified hpODN and dbODN, together with the natural
double-stranded control ODN (contODN). The natural double-
stranded 26-mer ODN (contODN) displayed a typically characteris-
tic B-DNA spectrum with a positive ellipticity at 273 nm, a negative
ellipticity at 248 nm, and a crossover between 252 and 258 nm
(Fig. 3).17 hpODN and dbODN possessing the thiol–maleamic acid
from equal equivalent of oligonucleotides C and D (10 lM) in
0.1 M MOPS buffer (pH 8.0) containing 1 M NaCl. After the incuba-
tion of dsODN at 37 °C for 10 h, the reaction product was moni-
tored by denaturing polyacrylamide gel electrophoresis (PAGE)
and further analyzed by matrix-assisted laser desorption/ioniza-
tion time-of-flight mass spectrometry (MALDI-TOF MS) after PAGE

1918
L. Cao, Z. Xi / Tetrahedron Letters 54 (2013) 1916–1920
Scheme 2. Synthesis of hpODN and dbODN.
methyl ester cross-linking at one or both ends of the helix
displayed an increase in both positive amplitude at 273 nm and
negative one, while there was no other significant change in the
overall CD spectrum. A similar CD characteristic was also observed
in double-stranded ODN possessing CuAAC bridges at the ter-
minis.2 The slight differences in the CD spectra between a natural
ODN and the cross-linked ODN may simply arise from inherent
differences of the sequences, or the increase in ellipticity at
273 nm may be related to an increased winding of the helix
induced by the presence of the dimmer b. The general consistency
of CD spectra suggested that the modifications on DNA terminis
did not significantly distort the helical geometry of the DNA
duplex.
dbODN were 72, and 85 °C, respectively, demonstrating signifi-
cantly enhanced thermal stability by the thiol–maleamic acid
methyl ester cross-linking of double-stranded DNA, in comparison
with the double-stranded ODN control (62 °C). These results of en-
hanced thermal stability by the thiol–maleamic acid methyl ester
cross-linking of double-stranded DNA were similar to those of a
CuAAC cross-linked 17-mer ODN (DTm 26.0 °C), although the
sequence and length of the ODN were different.2
The class II restriction endonuclease EcoRI recognizes the se-
quence 50-GAATTC and cleaves it between the G-4 and A-5.15 EcoRI
is sensitive to slight conformational changes and has been used to
probe for perturbations in the B-DNA structure; a reduction in the
rate of cleavage can be directly correlated to a change in helical
geometry. Therefore, analysis of the cleavage rates of hpODN and
dbODN can provide another way to determine whether our modi-
fications affect the double-strand structure of contODN. After
UV melting experiments were also conducted to evaluate the
thermal stability of the modified ODN via the thiol–maleamic acid
methyl ester cross-linking (Table 1). The Tm values for hpODN and

L. Cao, Z. Xi / Tetrahedron Letters 54 (2013) 1916–1920
1919
Figure 2. (a) Denaturing PAGE of the cross-linking reaction of hpODN and dbODN. Lanes 1, 2 showed ODN D, C, respectively (Scheme 1). Lanes 3, 4, 5, and 6 showed the cross-
linked reaction mixture of ODN D and C incubating for 0, 1, 2, and 10 h after annealing. Lanes 8, 9 showed the modified ODN H, G, respectively. Lane 10 is the reaction mixture
for dbODN after annealing. Lane 11 is the reaction mixture incubating at 37 °C for 5 h without annealing. Lane 7, 12 are DNA markers (50nt) with same sequence: GTACG
TACGT ACGTA CGTAC GTACG TACGT ACGTA CGTAC GTACG TACGT. (b) Denaturing PAGE of the EcoRI restriction enzyme digest. Lanes 13, 15, and 17 denoted contODN,
hpODN, and dbODN. Lanes 14, 16, and 18 showed the EcoRI restriction enzyme digest results of contODN, hpODN, and dbODN, respectively. (c) The time course curve for the
cross-linking reaction between ODN D and C. The yield of cross-linking at each time point was calculated from the gel scanning results by a BioRad Molecular Imager FX after
SYBR Gold staining on the gel.
180 min at 37 °C, hpODN and dbODN were almost completely
cleaved to produce digestion patterns unique for each sequence
(Fig. 2b). The fact that there is no loss in enzymatic activity for side
modification was consistent with the CD data presented above. It
can be concluded that thiol–maleamic acid methyl ester modifica-
tion on DNA ends does not significantly distort the B-DNA helical
geometry of the stem of the modified DNA.
In conclusion, a metal-free ‘click’ reaction: fast thiol–maleamic
acid methyl ester Michael addition, can be used not only in organic
synthesis, but also in oligonucleotide modification. A dimmer b
with two maleamic acid methyl ester groups can form facile cova-
lent ligation with thiol modified ODN at the 30- and 50-terminis
during annealing. The synthesized ODN were thermally stable
and the modification on DNA ends did not significantly disturb
the B-DNA helical geometry. This simple and fast intermolecular
end-capping method for cross-linking double stranded oligonucle-
otides with minimum modified stem may provide some advanta-
ges for bioactive application of nucleic acid, such as minimum
Figure 3. Circular dichroism spectra (conditions: 5 lM of ODN in sodium cacodyl-
ate–HCl buffer (1 mM, pH 7.0) containing 10 mM NaCl) of contODN, hpODN and
modified siRNA for RNAi,18 which will be reported in due course.
dsODN.
Acknowledgments
Table 1
This work was financially supported by the MOST
(2009ZX09503-022, 2010CB126102, 2011BAE06B05), and NSFC
(20932005).
Thermal stability of the thiol–maleamic acid methyl ester cross-
linked ODN
a
b
Tm (°C)
DTm (°C)
contODN
hpODN
dbODN
62
72
85
10
23
Supplementary data
a
Conditions: 1 lM each ODN, 1 mM sodium cacodylate–HCl
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.01.
112.
(pH 7.0), 1 mM NaC1.
b
Tm(modified) ꢀ Tm(control).

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L. Cao, Z. Xi / Tetrahedron Letters 54 (2013) 1916–1920
2009, 121, 4094–4097; (c) Lin, W.; Yuan, L.; Cao, Y.; Feng, Y.; Long, L. Chem. Eur.
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