Nucleic Acid Conformation Influences Postsynthetic Suzuki-Miyaura Labeling of Oligonucleotides

Article abstract of DOI:10.1021/acs.bioconjchem.0c00466

Chemoselective transformations that work under physiological conditions have emerged as powerful tools to label nucleic acids in cell-free and cellular environments. However, detailed studies investigating the influence of nucleic acid conformation on the

Full text of DOI:10.1021/acs.bioconjchem.0c00466

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Article
Nucleic Acid Conformation Influences Postsynthetic Suzuki−
Miyaura Labeling of Oligonucleotides
Manisha B. Walunj and Seergazhi G. Srivatsan*
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ABSTRACT: Chemoselective transformations that work under physiological conditions have emerged as powerful tools to label
nucleic acids in cell-free and cellular environments. However, detailed studies investigating the influence of nucleic acid
conformation on the performance of such chemoselective nucleic labeling methods are less explored. Given that nucleic acids adopt
complex structures, it is highly important to study the scope of the chemical modification method in the context of nucleic acid
conformations. Here we report a systematic study on the effect of local conformation on the postsynthetic Suzuki−Miyaura
functionalization of human telomeric (H-Telo) DNA repeat oligonucleotide (ON) sequences, which form multiple G-quadruplex
(GQ) structures. 5-Iodo-2′-deoxyuridine (IdU)-modified H-Telo ONs were synthesized by the solid-phase method, and when
subjected to Suzuki−Miyaura cross-coupling reaction, its efficiency was found to depend on the type of conformation and the
position of IdU label in different loops of the GQ structure. IdU-labeled GQs gave better yields as compared to single-stranded
random coil structures. However, the IdU-labeled duplex under different ionic conditions did not undergo the coupling reaction.
Further, using this method, we directly installed an environment-sensitive fluorescent probe, which photophysically reported the
formation as well as distinguished different GQ topologies of telomeric repeat. Collectively, this systematic study underscores the
influence of nucleic acid conformation, which has to be taken into account when establishing postsynthetic chemoselective
functionalization strategies.
orthogonal reactions. These reactions have provided modern
tools to (i) study nucleic acid localization, trafficking, and
profiling, (ii) develop diagnostic and therapeutic agents, and
(iii) develop functional nanomaterials.27−29 While many of
these benign chemoselective transformations have been
established with different nucleic acid sequences and under
different conditions including cell-free and cellular environ-
ments, the influence of nucleic acid conformation on the
performance of such chemoselective reactions has not been
well addressed. Such an effort will provide an understanding of
INTRODUCTION
■
Probing the structures of nucleic acids and their recognition
properties is highly important, and in this context,
oligonucleotides (ONs) labeled with biophysical probes greatly
facilitate the basic understanding of structure−function
relationships and also provide ways to develop diagnostic
and therapeutic tools.1−4 A technique, which has emerged in
the past decade as a very power tool for labeling nucleic acids
in vitro and in vivo is postsynthetic chemical labeling by using
chemoselective reactions.5−9 A reactive handle, which is
compatible under chemical or enzymatic labeling methods, is
introduced into ONs and further chemoselective reaction is
carried out to functionalize the ONs with the desired tag. Few
such reactions including azide−alkyne cycloaddition,10−18
Staudinger15,19−21 and inverse electron-demand Diels−Alder
(IEDDA)22−26 reactions have been demonstrated under
biologically benign conditions and hence termed as bio-
Received: August 19, 2020
Revised: October 4, 2020
https://dx.doi.org/10.1021/acs.bioconjchem.0c00466
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Figure 1. (A) Synthesis of 5-iodo-2′-deoxyuridine (IdU) phosphoramidite 3 for solid-phase synthesis of H-Telo DNA ONs. DMTr = 4,4′-
dimethoxytrityl, DMAP = 4-(dimethylamino)pyridine. (B) Flow diagram illustrating the synthesis of IdU (1)-labeled H-Telo DNA ONs, formation
of respective GQ structures and their influence on postsynthetic Suzuki−Miyaura cross-coupling reaction. Position of IdU label in the loop region
of each of the structures is shown in blue circle. The efficiency of Suzuki coupling to generate ONs labeled with an environment-sensitive probe
depended on the GQ conformation. PDB ID is given in parentheses.
the scope of a given postsynthetic reaction in modifying ONs,
as different nucleic acid conformations are likely to affect the
environment (e.g., steric, electronic, hydrophobic−hydrophilic
balance, etc.) around the reactive handle and hence its
reactivity with the cognate reaction partner.
can provide spectrum read-outs of a nucleic acid conformation
was not explored and is not straightforward.
Chemoselective reactions mostly result in the formation of
residual chemical groups (e.g., triazole), which however is not
a major problem in many bioconjugation strategies but is not
desirable when direct installation of biophysical probes is
required. In this context, palladium-mediated C−C bond
forming reactions have become popular in directly conjugating
probes onto biomacromolecules including DNA ONs.36−41 We
recently established a chemoselective posttranscriptional
functionalizing method to label RNA with biophysical probes
by using Suzuki−Miyaura reaction.42 This technique allowed
the direct synthesis of short RNA transcripts containing
fluorogenic and affinity tags from 5-iodouridine-modified
transcripts. Given the usefulness of Pd-mediated cross-coupling
reactions in functionalizing ONs, we sought to explore the
influence of nucleic acid conformational space on the efficiency
of postsynthetic cross-coupling reactions.
Here we report the investigation on the effect of nucleic acid
conformation on the efficiency of Suzuki−Miyaura coupling
reaction using polymorphic GQ structures as the study model.
5-Iodo-2′-deoxyuridine (IdU) phosphoramidite was incorpo-
rated into human telomeric (H-Telo) DNA ONs, which upon
annealing under different ionic conditions and in the presence
of a synthetic molecular crowding agent (PEG) adopted
different GQ topologies (Figure 1). Notably, the efficiency of
coupling reaction was found to depend on the type of GQ
conformation and the position of IdU in different loop regions.
However, the Suzuki reaction did not work on the duplex
structure of the telomeric DNA repeat. The environment-
sensitive fluorescent probe-labeled telomeric ONs thus
The photoreactivity of 5-halouracil has been used to probe
local DNA conformations such as A, B, and Z forms of the
DNA duplex and different topologies of G-quadruplexes
(GQ).30 UV irradiation of 5-halouracil-containing ONs results
in the formation of a uracil-5-yl radical, which in an atom-
specific and conformation-dependent manner induces hydro-
gen abstraction and intrastand cross-links.31 Further, ligand-
sensitized photochemical reactions with 5-bromouracil-modi-
fied GQ-forming ONs enabled the “photofootprinting” of GQ
folding topologies.32 Per se this is not a labeling approach, and
the end products including the deoxyribonolactone and side
reactions such as strand cleavage limit the downstream utility
of the ON products. GQ-forming sequences have also been
used as scaffolds for carrying out template-assisted azide−
alkyne cycloaddition reactions.33 In a similar approach, GQ
nanotemplates have been designed to facilitate regioselective
formation of triazole products via click reaction between a
series of alkyne and azide substrates.34 Only the substrates that
bind to a given GQ conformation undergo click reaction,
thereby offering a target-guided synthesis approach to discover
target-selective ligands. More recently, a chemoselective DNA
labeling method was established by reacting 5-vinyl-2′-
deoxyuridine-modified ONs with triazolinediones.35 This
reaction was found to be conformation-selective with the
GQ structure giving higher yields compared to duplex DNA.
However, the use of these reactions in attaching probes that
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Figure 2. Sequence of IdU-labeled H-Telo DNA ONs 4−17. The T residue in the first (4, 7, 10, 13), second (5, 8, 11, 14, 16), and third (6, 9, 12,
15, 17) loop of H-Telo DNA ONs was replaced with IdU. 18−20 correspond to unmodified ONs. C-rich ON 21 is complementary to DNA ONs
4−20.
Figure 3. (A and B) CD spectra of IdU-labeled ONs 12, 15, and 17 (solid lines) and control unmodified ONs 18−20 (dashed lines) in 50 mM
Tris-HCl buffer (pH 8.5) containing 100 mM KCl or 100 mM NaCl and 20% DMSO. Consistent with a literature report,47 ONs 15 and 19 in the
presence of Na+ ions did not give a CD profile corresponding to typical GQ structures (Figure 3B). (C and D) UV-thermal melting profiles of IdU-
labeled ONs 12, 15, and 17 (filled circles) and control unmodified ONs 18−20 (unfilled circles) under the above buffer conditions.
prepared faithfully reported the formation as well as
distinguished different GQ topologies.
adopts an antiparallel GQ structure, whereas in the presence of
K+ ions, it adopts mainly hybrid-type parallel−antiparallel
structures (hybrids 1 and 2).47 By slightly tweaking the 5′ and
3′ end nucleotides, one can also bias the repeat sequence to
predominantly adopt either hybrid 1 or hybrid 2 structure.48,49
In the presence of a synthetic crowding agent, polyethylene
glycol (PEG), the sequence adopts an all-parallel stranded GQ
structure, irrespective of the ionic conditions.50 Further, in
different GQ structures of H-Telo repeat, there are differences
in the conformations of the loop nucleotides (TTA). The loop
residues, especially T, replaced with an nucleoside analogue
probe photophysically reported the formation of different GQ
RESULTS AND DISCUSSION
■
Synthesis and Incorporation of IdU Phosphoramidite into
H-Telo DNA ONs. To evaluate the effect of nucleic acid
conformation on the efficiency of Suzuki reaction, we chose H-
Telo repeat sequence (TTAGGG)n as the study system for the
following reasons. H-Telo repeat sequence exhibits structural
polymorphism, as it can adopt multiple structures depending
on the ionic conditions, molecular crowding, and confined
environments (Figure 1B).43−46 In the presence of Na+ ions, it
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structures.51−53 Similarly, a dual-app probe composed of a
fluorophore and 19F label (fluorobenzofuran-modified nucleo-
side) placed in the loop position enabled two-channel
detection of different GQ forms by fluorescence and NMR
techniques.54 Taking a cue from these observations, we
decided to synthesize H-Telo DNA ONs labeled with IdU at
different loop positions to study postsynthetic functionalization
by Suzuki reaction.
5-Iodo-2′-deoxyuridine phosphoramidite 3 was synthesized
in simple steps as shown in Figure 1A.555-Iodo-2′-deoxyur-
idine 1 was protected with a DMTr group to obtain 5′-O-
DMT-protected 5-iodo-2′-deoxyuridine 2, which was reacted
with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite to
give phosphoramidite substrate 3 in good yields. A series of
ONs 4−17 containing IdU in different loop regions were
synthesized by using standard solid-phase synthesis protocol
(Figure 2). ONs were deprotected using ammonia at room
temperature for 48 h and then purified by polyacrylamide gel
electrophoresis (PAGE) using urea as the denaturant.
Deprotection at room temperature was necessary for
minimizing the deiodination of IdU-modified ONs. The purity
of ON products was analyzed by RP-HPLC, and their identity
was confirmed by mass measurement (Figures S1−S4 and
Table S1).
Table 1. Tm Values of IdU-Labeled and Control Unmodified
GQs in the Presence of KCl and NaCl
a
IdU-labeled ONs
Tm (°C)
control ONs
Tm (°C)
12 in KCl
15 in KCl
17 in KCl
12 in NaCl
15 in NaCl
17 in NaCl
70.5 0.5
59.3 0.5
66.8 0.7
51.3 0.6
47.7 0.9
56.6 0.8
18 in KCl
19 in KCl
20 in KCl
18 in NaCl
19 in NaCl
20 in NaCl
69.2 0.0
61.8 0.7
65.3 0.6
50.0 1.0
48.7 1.3*
56.2 0.8
a
In NaCl, ONs 15 and 19 showed a CD pattern, which did not
resemble a GQ topology (Figure 3B). However, the ONs in NaCl
showed a UV-thermal melting profile possibly for a random structure.
has been used for labeling protein and nucleic acids by Suzuki
and Sonogashira reactions.38,42,59,60 This catalytic system does
not require an inert atmosphere or degassed buffer to perform
coupling reactions. Benzofuran boronic acid (a) was preferred
as a cross-coupling partner because the resultant ON products
will be fluorescent and can sense GQ structures.51 The
reaction mixtures were analyzed by RP-HPLC, and the
fractions corresponding to the labeled ON products were
isolated and quantified (Figures S5−S9, Table 2). The identity
of the cross-coupled products were confirmed by MALDI-TOF
or ESI-MS analysis (Figures S10−S12 and Table S2).
IdU Labeling and Coupling Reaction Conditions Did
Not Affect the Formation and Stability of Respective
GQ Structures. Before subjecting the labeled ONs to
postsynthetic modification procedure, we first studied the
effect of IdU modification on the formation of native GQ
structures in coupling reaction conditions. As representative
examples, IdU-labeled (12, 15, and 17) and control
unmodified (18−20) H-Telo DNA ONs were annealed in
50 mM Tris-HCl buffer (pH 8.5) containing 100 mM KCl or
100 mM NaCl and 20% DMSO. The aqueous buffer used for
the Suzuki coupling reaction contained 20% DMSO to
solubilize the boronic acid/ester substrates. The CD spectra
of both control and modified H-Telo ONs under K+ ionic
conditions indicated the formation of hybrid-type GQ
structures matching the literature reports for the same
sequence (positive peak at ∼290 nm with a shoulder at
∼270 nm and a negative peak at 235−245 nm, Figure 3A).47
Likewise, in the presence of Na+ ions, the ONs (except for 15
and 19) produced a CD profile with a positive peak at ∼293
nm and a negative peak at ∼260 nm, indicating the formation
of an antiparallel GQ form (Figure 3B).47 Further, UV-thermal
melting analysis of control and modified H-Telo ONs under
different ionic conditions (NaCl and KCl) gave similar Tm
values, which were also in consensus with earlier reports
(Figure 3C and 3D, Table 1).56,57 Collectively, CD and Tm
data prove that the replacement of a thymidine residue with
IdU and Suzuki reaction conditions does not perturb the
folding and stability of the GQ structures.
All the ONs used in this study form a random coil structure
in the presence of LiCl and respective GQ structures in the
presence of NaCl and KCl.47,61 Reactions performed with IdU-
labeled G-quadruplex structures gave discernibly higher cross-
coupled ON products as compared to a random coil structure
formed by the same sequence (Figure 5, Table 2). GQ
structures containing an IdU label at the first dT position of
the first, second, and third loop (ONs 4−9) afforded the
coupled products in reasonably good yields. For example, in
NaCl, the antiparallel GQ topology gave similar yields when
IdU was placed in first (4), second (5), or third loop (6)
(Figure 5A). Similar results were observed in the case of hybrid
1 (ONs 4, 5, 6) and hybrid 2 (ONs 7, 8, 9) structures formed
in the presence of KCl (Figure 5A and 5B). On the other hand,
when the modification was placed at the second dT residue of
different loops (ONs 10−15), the reaction efficiency was
found to vary. A random structure of ONs 10−15 in the
presence of Li+ ions showed lower coupling efficiency with the
boronic acid substrate (Figure 5C and 5D). Interestingly, an
antiparallel GQ topology formed by ON 12 in which IdU is
present in the third loop gave 2- to 3-fold higher yields than
ONs 10 and 11 in which the modification is present in the first
and second loops, respectively (Figure 5C and Table 2). For
the same set of sequences in the presence of K+ ions, the
hybrid 1 form gave similar amounts of the coupled products
irrespective of the modification on different loops. However,
for reactions with hybrid 2-forming sequences 13−15, the
trend was reversed. Under Na+ ionic conditions, the ONs gave
similar amounts of the coupled products irrespective of the
position of modification, whereas in the presence of K+ ions
the ONs showed a difference in coupling efficiency (Figure
5D). There was a progressive increase in the product yield as
the iodo position was moved from the first (13) to the second
(14) and to the third loop (15).
Cross-Coupling Reaction Efficiency Depends on GQ
Conformation. H-Telo DNA ONs containing an IdU label in
the first and second T positions of different loops were
annealed in Tris-HCl buffer containing LiCl/NaCl/KCl and
reacted with benzofuran boronic acid (a) in the presence of
Pd(OAc)2L2 (Figure 4). Although, phosphine-based Pd
catalysts are useful in the postsynthetic modification of nucleic
acids in vitro and in cellular setting,36,37,58 they require an inert
atmosphere, degassed buffers, or oxygen scavengers to perform
the reaction. Therefore, we chose to use a catalytic system
made of Pd(OAc)2 and 2-aminopyrimidine-4,6-diol (L), which
Synthetic molecular crowding agents such as PEG can
induce G-rich sequences including human telomeric repeat to
adopt a parallel-stranded GQ topology.50 Labeled H-Telo ON
12 and control ON 18 were annealed in Tris-HCl buffer
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Figure 4. Postsynthetic Suzuki−Miyaura coupling reaction between IdU-labeled DNA ONs (4−17) and benzofuran boronic acid substrate a.
Tris-HCl buffer (pH = 8.5) containing either 100 mM NaCl or
100 mM KCl. Both control (18·21, 19·21, 20·21) and
modified DNA duplexes (12·21, 15·21, 17·21) showed similar
CD profiles and were characteristic of a B form of duplex
(Figure S16).63 Interestingly, the duplexes formed using IdU-
modified ONs were found to be completely unreactive under
Suzuki reaction conditions even after 12 h of incubation
(Figure S17). This observation further suggests the influence
of conformation on the coupling reaction efficiency.
Change in Ionic Conditions Does Not Influence the
Coupling Efficiency. It is reported that inorganic salts alter
the efficiency of organic reactions such as Suzuki−Miyaura
cross-coupling, Diels−Alder cycloaddition, Wittig reactions, to
name a few.64 To test the influence of added salts on the
postsynthetic Suzuki−Miyaura cross-coupling, a short 5-
iodouridine-modified ON 22, which does not form any
secondary structure, was reacted with benzofuran boronic
acid in the presence of LiCl/NaCl/KCl (Scheme S1). Different
ionic conditions did not affect the reaction efficiency of the
unstructured ON, indicating that the differences in reaction
efficiency of GQs, random coil, and duplexes are due to the
conformation adopted by ONs and not due to added salts
(Figure S18 and Table S3).
Possible Reasons for the Differences in the Reactivity
of the ONs. Iodo modification at the C5 position of the
nucleoside incorporated into DNA duplexes should be present
in the major groove, which is wide but is also deep. Due to this
constraint in space, it is likely that the Pd(OAc)2L2 catalytic
system and/or the intermediates in the coupling cycle may not
attain the correct geometry for the coupling process. This
restriction is somewhat released in the random coil structures,
and hence we observed reasonable coupling efficiency.
The H-Telo ONs form GQs with three loops formed by
TTA bases. IdU-modified GQs in general produced higher
yields of the coupled ON products as compared to the random
coil structure. When IdU was placed at the first dT in each of
the three loops, the coupling efficiency was found to be the
best, irrespective of the GQ topologies. On the basis of the 3D
structures of antiparallel and hybrid GQs,48,65,66 the first dT in
all loops though show subtle differences in their interaction
with neighboring bases, they are exposed, and hence the C5
iodo label is accessible for the coupling reaction (Figures S19
and S20). On the other hand, IdU placed at the second dT
position in all the three loops reacted less efficiently with the
boronic acid substrate. In the antiparallel form, the second dT
Table 2. Isolated Yields of Cross-Coupled Products (4a−
15a) Obtained by the Suzuki−Miyaura Reaction with ONs
Containing IdU in Different Loops and under Different
a
Ionic Conditions (Li+/Na+/K+)
isolated yields of cross-coupled
product (%)
IdU (1)-labeled
H-Telo DNA ONs in
structures
formed by ONs first loop
second
loop
third
loop
different salts
b
Li+(1st T)
random coil
antiparallel
hybrid type 1 46 (4a)
random coil
random coil
hybrid type 2 43 (7a)
36 (4a)
58 (4a)
33 (5a)
52 (5a)
62 (5a)
16 (8a)
32 (8a)
46 (8a)
28 (6a)
54 (6a)
55 (6a)
31 (9a)
55 (9a)
48 (9a)
b
Na+(1st T)
b
K+(1st T)
Li+(1st T)
b
17 (7a)
42 (7a)
b
Na+(1st T)
b
K+(1st T)
c
Li+(2nd T)
random coil
antiparallel
13 (10a) 11 (11a) 16 (12a)
10 (10a) 18 (11a) 33 (12a)
c
Na+(2nd T)
c
K+(2nd T)
hybrid type 1 21 (10a) 20 (11a) 22 (12a)
c
Li+(2nd T)
random coil
5 (13a)
15 (13a) 19 (14a) 15 (15a)
hybrid type 2 9 (13a) 15 (14a) 23 (15a)
8 (14a)
7 (15a)
c
Na+(2nd T)
random coil
c
K+(2nd T)
a
All reactions were performed on a 2.5 nmol scale of IdU-labeled
b
DNA ONs (4−15). IdU (1) is placed at the first dT position of
different loops formed by TTA nucleotides. IdU (1) is placed at the
c
second dT position of different loops formed by TTA nucleotides.
containing 40% PEG (200) and 100 mM KCl. Consistent with
earlier reports, CD profiles of the ONs in PEG confirmed the
formation of a parallel GQ structure (Figure S14A). Suzuki
reaction between the parallel form of 12 and benzofuran
boronic acid in the presence of Pd(OAc)2L2 catalyst did not
produce any detectable coupled product (Figure S14B). Closer
examination of the HPLC chromatogram revealed consid-
erable reduction in the peak intensity of the Pd catalyst
(retention time ∼3.7 min), which could be due to the
sequestering effect of PEG. To ascertain this, reactions were
performed at different percents of PEG (5−30%, Figure S15).
The formation of cross-coupled product 12a decreased with
increase in the % of PEG in the reaction mixture. Higher
amounts of PEG could decrease the diffusion rate and also
could coordinate with palladium, thereby reducing the effective
concentration of the catalyst.62
Duplexes Made of IdU-Labeled H-Telo DNA ONs Did
Not Undergo the Coupling Reaction. H-Telo DNA ONs
were hybridized to complementary C-rich DNA ON 21 in
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Figure 5. Bar diagram of isolated yields of cross-coupled products (4a−15a) obtained from reactions between IdU-labeled ONs (4−15) and
benzofuran boronic acid substrate a. Reactions with ONs (A) 4, 5, and 6; (B) 7, 8, and 9; (C) 10, 11, and 12; (D) 13, 14, and 15 in the presence
of different ions. See Figure S13 for a representative bar diagram showing the errors in the isolated yields.
in the first loop is almost buried and stacks on the G-tetrad
core (Figure S19A). In the second loop the dT is stacked from
one side with adjacent dA (Figure S19B), and in the third loop
it is not stacked but is located in the groove wherein there is a
void space around the C5 position (Figure S19C). Similarly, in
the hybrid structure the second dT is either inaccessible or
stacked over the G-tetrad (Figure S20). Hence, the differences
in reactivity exhibited by the ONs is likely due to steric effects
and differences in the interaction of the IdU label with adjacent
nucleobases originating from the nucleic acid conformation.
Benzofuran Modification Introduced by Postsyn-
thetic Suzuki Reaction Fluorescently Distinguishes
Different GQ Topologies. To evaluate whether the modified
ONs synthesized by Pd-catalyzed reactions are suitable for
downstream biophysical analysis, benzofuran-modified H-Telo
DNA ON 16a was subjected to fluorescence analysis. ON 16a
containing the label at the first dT base of the second loop
formed respective GQ topologies in different ionic conditions
without hampering the native fold, which was confirmed by
CD analysis (Figure S21). We then recorded the fluorescence
spectra of the ON in the presence of K+ or Na+ ions by exciting
the samples at 330 nm. The duplexes made of ON 16a in KCl
and NaCl exhibited very low fluorescence (Figure 6). The
mixed hybrid-type GQs (hybrids 1 and 2, blue line) formed by
16a in KCl exhibited ∼6-fold enhancement in fluorescence
intensity with a red shift in emission maximum (λem = 434 nm)
as compared to the duplex form (λem for 16a·21 is 427 nm).
Antiparallel topology displayed further enhancement in
fluorescence intensity (∼14-fold, black line) compared to its
duplex form. The probe also distinguished between hybrid 1
Figure 6. Fluorescence spectra of GQs of H-Telo DNA ON 16a and
corresponding duplexes under different ionic conditions. Spectra of
GQs of ONs 5a (hybrid 1), 8a (hybrid 2), and 16a (mixed hybrids)
in KCl. Samples (0.5 μM) were excited at 330 nm with an excitation
and emission slit width of 5 and 6 nm, respectively.
and hybrid 2 forms (Figure 6). ON 5a, which predominantly
forms the hybrid 1 structure in KCl, displayed an intense
emission band (magenta line), nearly 5-fold higher than that of
the hybrid 2 structure formed by ON 8a (red line). ON 16a, as
before, showed intermediate fluorescence, as this sequence
forms both hybrid 1 and 2 forms in KCl (blue line). The ability
of the probe to fluorescently distinguish different GQ
topologies is due to differences in the microenvironment of
the probe in different conformations.51,52 Collectively, these
results clearly indicate the usefulness of Pd-catalyzed cross-
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coupling reactions in constructing ONs labeled with functional
probes.
ON 21 (8.8 μM) in Tris-HCl buffer (50 mM, pH 8.5)
containing either 100 mM NaCl or 100 mM KCl and 20%
DMSO. All the samples were heated at 90 °C for 3 min. The
samples were slowly cooled to RT and kept on an ice bath for
at least 1 h. The CD spectrum was recorded from 200 to 350
nm on a J-815 CD spectropolarimeter (Jasco, Portland, OR)
using 1 nm bandwidth at 20 °C. Each CD profile is an average
of three scans collected at a scan speed of 100 nm/min. All CD
measurements were carried out in duplicate, and the spectra
were corrected using an appropriate buffer solution without
DNA ONs.
UV−Thermal Denaturation Experiments. The samples
prepared for CD analysis were diluted with respective 1X
buffer such that the final concentration of the ON samples was
1 μM. Thermal melting analysis was performed in duplicate by
using a Cary 300 Bio UV−vis spectrophotometer. The
temperature was increased from 20 °C to 90 °C at 1 °C/
min, and the absorbance was measured every 1 °C interval at
295 nm. Both forward and reverse cycles were used for the Tm
determination.
Postsynthetic Suzuki−Miyaura Reactions. Reactions
with IdU-Labeled DNA ONs 4−17. Samples of IdU-labeled
DNA ONs 4−17 (2.5 nmol, 50 μM, 1 equiv) in Tris-HCl
buffer (50 mM, pH 8.5) containing 100 mM salt (LiCl, NaCl,
or KCl) and 20% DMSO were annealed to form the respective
DNA structures by heating at 90 °C for 3 min. The samples
were cooled to attain RT and placed in an ice bath for at least 1
h. Further, benzofuran boronic acid a (2.5 mM, 50 equiv) was
added to the above solution, and the reaction was initiated by
adding catalyst Pd(OAc)2L2(0.1 mM, 2 equiv). The final
volume of the reaction was 50 μL, and the overall percentage
of DMSO was kept at 20% v/v. All reactions were performed at
37 °C without degassing the buffer.
Reactions with IdU-Labeled DNA Duplexes. IdU-modified
DNA ONs 12, 15 and 17 (2.5 nmol, 50 μM, 1 equiv) and
complementary C-rich DNA ON 21 (2.75 nmol, 55 μM, 1.1
equiv) in Tris-HCl buffer (50 mM, pH 8.5) containing either
NaCl or KCl (100 mM) and 20% DMSO were annealed to
form duplexes as mentioned above. Boronic acid substrate a
(2.5 mM, 50 equiv) and Pd(OAc)2L2(0.1 mM, 2 equiv) were
added to a final volume of 50 μL. All reactions were performed
at 37 °C without degassing the buffer.
Analysis of the Reaction. The reaction mixtures were
filtered using spin filters (0.45 μm pore size), and the filters
were washed with 50 μL of autoclaved water. The filtrates were
analyzed by RP-HPLC (Phenomenex-Luna C18 column, 250
× 4.6 mm, 5 μm). Mobile phase A: 50 mM TEAA buffer (pH
7.0), mobile phase B: acetonitrile. Flow rate: 1 mL/min.
Gradient: 0−30% B in 35 min, 30−100% B in 10 min and
100% B for 5 min. The run was monitored at 260 and 330 nm
(for the benzofuran coupled ON products). The fractions
corresponding to the products were collected and lyophilized
at least three times to remove the volatile TEAA buffer. The
ON products were quantified by UV absorption, and their
identity was confirmed by mass analysis. See SI for HPLC
profiles and mass data.
Detection of GQ Structures by Fluorescence. H-Telo
ONs 5a/8a/16a (0.5 μM) were annealed at 90 °C for 3 min in
10 mM Tris−HCl buffer (pH 7.5) containing either 100 mM
KCl or 100 mM NaCl. To obtain a DNA duplex (16a·21), ON
16a and complementary C-rich ON 21 were assembled by
heating a 1:1.1 mixture in 10 mM Tris-HCl buffer (pH 7.5)
containing either 100 mM KCl or 100 mM NaCl at 90 °C for 3
Reaction yields of this conformation-selective modification
and fluorescence properties of the ON products cannot be
easily correlated, as factors that influence yields and
fluorescence outcome could be different and also would
depend on the boronic acid substrate. However, in our case we
did see a correlation between the duplex form and the G-
quadruplex structures. While the iodo-modified duplex did not
yield any coupled product with benzofuran boronic acid, iodo-
modified GQ structures were found to be suitable for
postsynthetic modification by Suzuki reaction. Further,
benzofuran-modified GQs exhibited significantly higher
fluorescence as compared to very weak fluorescence exhibited
by the benzofuran-modified duplex under different ionic
conditions (Figure 6).
CONCLUSIONS
■
We successfully performed postsynthetic Suzuki−Miyaura
cross-coupling reactions on various DNA conformations such
as random coil, GQ, and duplex structures. The differences in
reactivity displayed by various DNA conformations of the
human telomeric repeat ON sequences mainly depended on
the position of the IdU label in the loop, and the electronic and
steric environment around IdU. The results indicate that this
Suzuki-based ON functionalization method is conformation-
dependent, as the efficiency of the reaction decreased in the
following order: GQ topology > single-stranded random coil
and no reaction with duplex DNA. Further, this approach is
modular, as various functional tags can be directly installed by
reacting boronic acid/ester substrates with easily accessible
IdU/IU-labeled ONs. Here we synthesized telomeric repeats
labeled with an environment-sensitive fluorescent probe, which
faithfully reported the formation as well as distinguished
different GQ structures via changes in its emission intensity
and maximum. Taken together, this study highlights the
importance of nucleic acid conformation, which needs to be
taken into consideration when establishing postsynthetic
functionalization strategies based on chemoselective reactions.
EXPERIMENTAL SECTION
■
Synthesis and characterization data of 5-iodo-2′-deoxyuridine
phosphoramidite substrate 3 are given in Supporting
Information. Detailed procedure for the incorporation of 3
into H-Telo DNA ON sequences by the solid-phase method,
HPLC traces, and characterization by MALDI-TOF and ESI
mass spectroscopy techniques are provided in Supporting
Information. HPLC chromatograms of Suzuki−Miyaura cross-
coupling reactions and mass spectra of cross-coupled products
are also given in Supporting Information.
CD Measurements. Respective GQ structures were
formed by annealing IdU-labeled DNA ONs 12/15/17 (8
μM) and control unmodified H-Telo DNA ONs 18−20 (8
μM) in Tris-HCl buffer (50 mM, pH 8.5) containing either
100 mM NaCl or 100 mM KCl and 20% DMSO. To obtain a
parallel conformation, IdU-modified ON 12 and unmodified
ON 18 were annealed in 50 mM Tris−HCl buffer (pH 8.5)
containing 40% PEG 200, 100 mM KCl, and 20% DMSO. The
corresponding IdU-modified (12•21, 15·21, 17·21) and
unmodified DNA duplexes (18•21, 19·21, 20·21) were
prepared by heating a 1:1.1 mixture of H-Telo DNA ONs
12/15/17/18−20 (8 μM) and complementary C-rich DNA
G
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pubs.acs.org/bc
Article
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min. All the samples were then cooled slowly to attain RT and
placed in an ice bath for 1 h. Each sample was excited at 330
nm with excitation and emission slit widths of 5 and 6 nm,
respectively. All fluorescence experiments were performed in
triplicate with appropriate blank corrections in a micro
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Horiba Jobin Yvon, Fluoromax-4 at 20 °C.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.bioconjchem.0c00466.
■
sı
Experimental details, characterization data, and support-
ing figures and tables (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Seergazhi G. Srivatsan − Department of Chemistry, Indian
Institute of Science Education and Research (IISER), Pune
411008, India; orcid.org/0000-0001-5765-3967;
Email: srivatsan@iiserpune.ac.in
Author
Manisha B. Walunj − Department of Chemistry, Indian Institute
of Science Education and Research (IISER), Pune 411008,
India
(16) Nguyen, K., Fazio, M., Kubota, M., Nainar, S., Feng, C., Li, X.,
Atwood, S. X., Bredy, T. W., and Spitale, R. C. (2017) Cell-selective
bioorthogonal metabolic labeling of RNA. J. Am. Chem. Soc. 139,
2148−2151.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.bioconjchem.0c00466
̈
(17) Winz, M.-L., Linder, E. C., Becker, J., and Jaschke, A. (2018)
Site-specific one-pot triple click labeling for DNA and RNA. Chem.
Commun. 54, 11781−11784.
Notes
(18) Krell, K., Harijan, D., Ganz, D., Doll, L., and Wagenknecht, H.-
A. (2020) Postsynthetic modifications of DNA and RNA by means of
copper free cycloadditions as bioorthogonal reactions. Bioconjugate
Chem. 31, 990−1011.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
M.B.W. is grateful to CSIR, India, and Wellcome Trust-DBT
India Alliance for a graduate research fellowship. Our thanks to
Rupam Bhattacharjee for his help in the mass analysis of ONs.
This work was supported by a Wellcome Trust-DBT India
Alliance senior fellowship (IA/S/16/1/502360) to S.G.S.,
which is highly appreciated.
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