Effect of preorganization on the affinity of synthetic DNA binding motifs for nucleotide ligands

Article abstract of DOI:10.1039/c5ob00508f

Triplexes with a gap in the purine strand have been shown to bind adenosine or guanosine derivatives through a combination of Watson-Crick and Hoogsteen base pairing. Rigidifying the binding site should be advantageous for affinity. Here we report that cl

Full text of DOI:10.1039/c5ob00508f

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Biomolecular Chemistry
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Effect of preorganization on the affinity of
synthetic DNA binding motifs for nucleotide
ligands†
Cite this: Org. Biomol. Chem., 2015,
13, 5734
S. Vollmer and C. Richert*
Triplexes with a gap in the purine strand have been shown to bind adenosine or guanosine derivatives
through a combination of Watson–Crick and Hoogsteen base pairing. Rigidifying the binding site should
be advantageous for affinity. Here we report that clamps delimiting the binding site have a modest effect
on affinity, while bridging the gap of the purine strand can strongly increase affinity for ATP, cAMP, and
FAD. The lowest dissociation constants were measured for two-strand triple helical motifs with a propy-
lene bridge or an abasic nucleoside analog, with Kd values as low as 30 nM for cAMP in the latter case.
Taken together, our data suggest that improving preorganization through covalent bridges increases the
affinity for nucleotide ligands. But, a bulky bridge may also block one of two alternative binding modes for
the adenine base. The results may help to design new receptors, switches, or storage motifs for purine-
containing ligands.
Received 16th March 2015,
Accepted 15th April 2015
DOI: 10.1039/c5ob00508f
www.rsc.org/obc
molar range. Another class of binders are nucleic acid-based
binding motifs. Best known are aptamers, i.e. RNA sequences
Introduction
Nucleoside phosphates are key building blocks of the cell. Tri- identified from sequence libraries by systematic evolution by
phosphates are the monomers for both DNA and RNA syn- exponential enrichment (SELEX).13 Aptamers for a number of
thesis.1 Adenosine monophosphate is also part of the nucleoside phosphates or cofactors, including ATP,14,15
structure of several cofactors, including acetyl-CoA, nicotin- cAMP,16 SAM,17 FAD,18,19 and GTP20 are known, and so are
amide adenine dinucleotide (NADH/NAD+), flavin adenine ribonucleopeptides21,22 that bind ATP or GTP.
dinucleotide (FADH2/FAD), and S-adenosylmethionine (SAM).
Recently, designed nucleic acid-based binders for purines
Further, cyclic anhydrides of purine nucleotides are second were reported that feature a gap in the oligopurine strand of a
messengers, including 3′,5′-cycloadenosine monophosphate triplex as binding site.23–25 The designed binding motifs show
(cAMP) and 3′,5′-cycloguanosine monophosphate (cGMP), with low micromolar affinity for biologically important nucleoside
important functions in intracellular signal transmission and phosphates and cofactors and bind their targets through a
amplification.2 Binding nucleoside phosphates with high combination of Watson–Crick and Hoogsteen base pairing
affinity and selectivity is therefore critical, and nucleotide (Fig. 1). They can be immobilized on porous supports, leading
binders are a focus of studies in analytical chemistry,3,4 medi- to macroscopic storage devices that can reversibly bind cofac-
cine,5 and chemical biology.6
tors, so that cofactor-dependent biochemical transformations
Synthetic nucleotide binders have been constructed from a can be switched on or off through modest temperature
number of different compound classes. Among them are mole- changes.24 The selectivity for different cofactors can be tuned
cular tweezers,7 scorpiand receptors,8 uranyl-salophen recep- through small changes in the sequence, and affinity for non-
tors,9 pyridinium tripods,10 copper complexes,11 and nucleosidic cofactors, such as folic acid can be induced.24 An
phenylenediamine receptors.12 Most of these purely synthetic RNA version of a triplex-based motif has been expressed in live
binders give dissociation constants in the millimolar to micro- cells, where it acted as a sink for cGMP, a secondary messen-
ger.25 Triplex-based motifs have also been used for the con-
struction of fluorescence sensors for several purines using
fluorophores covalently linked to nucleobases of one of the
triplex strands.26,27
Pairing between oligonucleotides is known to be
accompanied by strong enthalpy-entropy compensation.28 The
large entropic penalty for duplex formation can be reduced by
rigidifying the strands involved. The most common way of
Institute for Organic Chemistry, University of Stuttgart, 70569 Stuttgart, Germany.
E-mail: lehrstuhl-2@oc.uni-stuttgart.de; Fax: +49 (0) 711 608 64321;
Tel: +49 (0) 711 608 64311
†Electronic supplementary information (ESI) available: MALDI-TOF mass
spectra of modified oligonucleotides, additional UV/VIS-spectra from filtration
assays, HPLC traces, UV-melting points, and additional UV-melting curves. See
DOI: 10.1039/c5ob00508f
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was expected to labilize triplexes, further motivating our
search for ways to rigidify the gap-containing, nucleotide-
binding triplex structures. Here we report the synthesis of
oligonucleotides that form triplex-based binding motifs with a
clamp flanking the binding site (II) or a bridge connecting the
termini of the oligopurine strands (III, Fig. 1b).
Either type of construct was found to form stable triplexes,
as determined by UV-melting analysis and to bind cofactors,
such as SAM, FAD, ATP, or cAMP, as determined by equili-
brium filtration. Depending on the design of the oligonucleo-
tides, micro- to nanomolar dissociation constants were found,
with the tightest binding in the case of bridged triplexes and
cAMP as ligand, and the weakest binding in the case of ATP.
Results
Scheme 1 shows the oligonucleotides synthesized to form
triple helices of general structure II. The group of strands
included all-pyrimidine strands 1–5 that can form an intermole-
cular triplex, and intramolecular folding motif 6 that features
both the two oligopyrimidines and the oligopurine segment of
a parallel triplex. The strands were prepared by automated
DNA synthesis, starting from controlled pore glass (cpg)
loaded with the 3′-terminal nucleoside, using a combination
of conventional phosphoramidites and building blocks syn-
thesized according to slight modifications of literature proto-
cols. Compound 1, which features a T5 clamp, lacks modified
residues and was purchased from a commercial source. Oligo-
nucleotide 2 contains a hexaethylene glycol (HEG) linker as
clamp and was prepared with phosphoramidite 8 as unnatural
building block. The loop serving as clamp in 3 is a combi-
nation of a T3 segment and the pyrenyl C-nucleoside (Py),
incorporated by employing phosphoramidite 9, prepared as
Fig. 1 Binding adenosine through a combination of Watson–Crick and
Hoogsteen base pairing. (a) Base pairing interactions, (b) strands
arrangements in triple helices without (I), with a clamp (II), or with a
bridge (III).
achieving this is by clamping down on conformational flexi- previously reported.41,42 The pyrenyl residue can be expected
bility through an additional covalent linkage. Locked nucleic to stack on the termini.41,43,44 Finally, the largest and presum-
acids (LNAs) are one well-known example for this, relying on ably most strongly preorganizing clamp was incorporated in 5
bridges between the 2′- and 4′-positions of the riboses.29,30 and 6 by using the perylene diimide building block 10.39,40
Alternatively, two nucleotides may be bridged with a synthetic Of the two strands, all-pyrimidine sequence 5 has a larger
lock. For example, intrastrand locks were recently shown to number of deoxycytidine residues, as compared to 1–4. The
increase the affinity and base pairing selectivity of DNA probes triple helical binding motif 6 was expected to form the desired
binding RNA target strands.31 Interstrand crosslinking with triplex helical structure through intramolecular folding, with
disulfide bridges,32 metal-salen complexes,33 or triazolides34 the PDI residue again designed to act as clamp sealing the
as bridges can strongly stabilize duplexes. When the synthetic binding site for adenosine-containing ligands. An entirely
lock is at the termini of two complementary strands, hairpins intramolecular triple helical motif is better suited for immobili-
are formed. Here, the lock may be constructed from a stilbene zation and thermal capture/release cycles without loss of
diether35,36 or perylene diimide.37 Clamped constructs are also strands than a bimolecular system formed by hybridization of
known for parallel DNA triplexes.38 Typically, the two homo- unlinked strands.24
pyrimidine strands of a triplex are linked by a naphthalene
diimide or perylene diimide clamp, leading to increases in characterized by MALDI-TOF mass spectrometry. Overall yields
free energy of formation of up to −12.3 kcal mol−1 39,40
of the oligonucleotide syntheses ranged from 3–23%, a range
All six oligonucleotides (1–6) were HPLC purified and
.
It was therefore interesting to ask whether covalent locks in typical for custom-synthesized, modified DNA strands. Fig. 2
the structure of triplex-based binders for nucleotides improve shows a representative spectrum. Other spectra can be found
pairing properties and thus target affinity. Since known triplex- in the ESI (Fig. S8–S15†).
based DNA motifs are at least 50 nucleotides in size, it was
Fig. 3 shows triple helical structures expected to form upon
also desirable to prune them. The shortening of the strands hybridizing 1–5 with target strands 11 or 12. Either of the
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Scheme 1 Synthesis of oligodeoxynucleotides 1–6.
Fig. 4 Representative UV/VIS-spectra from binding assay. Spectra of
FAD solutions after exposure to 6 and filtration. The bold line is the
spectrum of the reference filtration, in the absence of oligonucleotides,
whereas the dotted line is from the sample containing both DNA triplex
and ligand. Conditions: 10 µM DNA, 10 µM FAD, 10 mM phosphate
buffer (pH = 6), 1 M NaCl; centrifugation at 4 °C, using filters with a
3 kDa size cut-off.
Fig. 2 MALDI-TOF spectrum of 6 (linear negative mode).
caused by the hairpin facilitated separation of ligand and oli-
gonucleotide motif in filtration assays (vide infra). The
sequence of the hairpin segment was chosen to be GC-rich, in
order to prevent cross hybridization with the TA-rich triple
helical segment. Finally, triple helices 5 : 12 and 6 present a
slightly different electrostatic profile, compared to 1–4 : 11, due
to the additional CGC+ base triplet.
For all six designs, the formation of triplexes was induced
by thermal hybridization, and UV-melting curves showed
melting points in the range of 14–42 °C, that is above the
temperature at which binding of ligands was measured (4 °C).
The ability of the triple helices to bind cAMP, SAM or FAD was
tested by membrane filtration, following a protocol previously
described.23,24 Binding constants were determined by calcu-
lation of the fraction of bound ligands from the concentration
found in the filtrate, as compared to that of a control filtration
without oligonucleotide motifs. A representative set of UV/VIS-
spectra for FAD is shown in Fig. 4. The dissociation constants
calculated from the fractions of bound and unbound ligand
are given in Table 1.
Fig. 3 Triple-helices formed between 1–5 and oligopurine targets 11
or 12, or by intramolecular folding (6). Ligands are shown in red for
clarity. See Scheme 1 for the structure of clamp residues.
purine-rich strands contained a hairpin sequence to minimize
frame-shifting that leads to alternative structures, with the
ligand binding site blocked. Further, the increase in size
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Table 1 Dissociation constants (Kd)a for complexes of triple helices
with cAMP, SAM, or FAD as ligands
Sequences
Clamp
Kd (cAMP)
Kd (SAM)
Kd (FAD)
1 : 11
2 : 11
3 : 11
4 : 11
5 : 12
6
T5
HEG
Py
PDI
PDI
PDI
>150
90
60
40
>150
80
>150
>150
80
50
98
>150
>150
>150
9
8
8
34
a As determined by equilibrium filtration; conditions: 10 µM DNA
strands, 10 µM ligand, 10 mM phosphate buffer (pH = 6), 1 M NaCl,
4 °C.
Triple helical system 1 : 11 showed no detectable binding of
either of the three ligands in our assay, with dissociation
constants >150 µM (Table 1). Possibly, the flexible T5-clamp
does not offer a sufficient level of preorganization at the
binding site, and insufficient stacking interactions for incom-
ing adenosine phosphates. Bimolecular complex 2 : 11, with its
shorter linker, known from similar DNA helices,45 but limited
stacking capabilities, gave detectable binding for cAMP, but no
measurable affinity for SAM or FAD. Pyrene-containing 3
formed a triplex with 11 that gave detectable affinity for cAMP
and SAM, suggesting that its aromatic stacking moieties did
attract adenosine phosphates more readily than the triplexes
formed by either 1 or 2. Only when the large aromatic ring
system of the perylene diimide was offered as clamp structure
(4 : 11), did significant binding occur for any of the three
ligands. Here, for FAD, with its large flavin residue, a low
micromolar Kd was measured.
Perylenes have previously been reported to be excellent
stacking partners for base triplets.39 Triplex 5 : 12, with its less
stable triplex gave lower affinity for both cAMP and SAM,
suggesting that preorganization is indeed important for
binding nucleotide ligands tightly. Finally, 6 which forms an
intramolecular triplex, showed strong affinity for all three
cofactors tested, with Kd values in the range of 8–80 µM.
Neither of the ligands induced a shift in the melting point,
though, when added to the solution of the triplexes of this
group.
The results obtained for 1–6 indicated that the affinity for
ligands can be tuned by the choice of the clamp. But, it was
also evident that for the clamp-containing triple helices, the
affinity for cAMP, FAD and SAM was lower than for triplexes
with a binding site in the interior of a triple helix,23–25 rather
than at its terminus. This made us turn to binding motifs of
general structure III (Fig. 1) and designs that feature a bridge
between the oligopurine strands of the central strand. The
bimolecular triple-helical structures shown in Fig. 5 were
therefore pursued next. This included 13 and 14, which feature
hexa- and tetranucleotide loops similar to those described in
earlier work on folding motifs than can be expressed in cells.25
Shorter loops had proven poor bridges in our earlier studies,25
possibly because of conformations that interfere with binding.
Fig. 5 Triple-helices between bridged oligopurines (13–18) and oligo-
pyrimidine 19.
Also included in the group of bridged oligopurines were 15
and 16 that feature acyclic chains with a long (HEG)45 or a
short linear chain (C3).26,27 The latter has the same number of
carbon atoms between two phosphates as a natural ribose
(shortest path along the backbone). Presumably the most rigid
bridge is that of 17, with an abasic site analog (AS) replacing a
natural deoxynucleoside residue. Abasic sites have been tested
before in duplexes,46–48 but, to the best of our knowledge, not
in triplex binding motifs. In 17, the AS-bridge leaves space for
the nucleobase only. It occupies the space usually taken up by
the ribose phosphate of the ligand, at least, if the ligand was
to bind in the same orientation as the deoxyadenosine of an
uninterrupted oligopurine chain. Finally, oligopurine 18,
which features an uninterrupted strand was included as
control compound, forming triplexes that do not contain a
binding site.
For all but one of the six different triplex structures, the
triplex-to-duplex and duplex-to-single strands transitions
coincided, so that a single sigmoidal curve was observed, even
in the absence of ligands (see the left-most curve in Fig. 6a).
Only for the triple helix 14 : 19 was the UV-melting point for
the dissociation of the triplex-forming segment low enough to
give a discernible second transition (see Fig. S19, ESI†). Three
different adenosine phosphates were tested as ligands, namely
cAMP, FAD, and ATP. The triphosphate was included because
it should be a weak ligand, due to the electrostatic repulsion
between the DNA backbones and the oligophosphate moiety.
In the case of cAMP and 17 : 19, the ligand induced a signifi-
cant shift in the UV-melting point (Fig. 6a and Table 2),
suggesting that the ligand was bound so tightly that it
remained in the binding pocket up to the temperature where
the triplex-forming segment dissociated from the remaining
duplex. This phenomenon has been observed previously for
very stable complexes with triple helical binding motifs.23,24
Triplex 16 : 19 with the propylene bridge showed a similar
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Table 3 Dissociation constants (Kd) [µM] for complexes of 13–18 and
19 and ATP, cAMP, FAD, or cGMP as control compounda
Sequences
Bridge
ATP
cAMP
FAD
cGMP
13 : 19
14 : 19
15 : 19
16 : 19
17 : 19
18 : 19
T6
T4
HEG
C3
AS
A
>150
129
29
2.8
5
20
8
2.8
0.04
0.03
>150
16
10
15
1.4
1.8
>150
>150
>150
>150
>150
>150
>150
>150
a As determined by equilibrium filtration; conditions: 10 µM DNA
strands, 10 µM ligand, 10 mM phosphate buffer (pH = 6), 1 M NaCl,
4 °C.
High affinity for adenosine phosphate ligands was also
found in binding assays based on equilibrium filtration
(Table 3). The group of nucleotides assayed now also included
cGMP as control compound that offers a stacking surface
similar to that of cAMP, but a different hydrogen bonding
pattern, so as to test for the specificity of base pairing. A pair
of representative absorption spectra from the binding assay
involving 17 : 19 and cAMP are shown in Fig. 6b. It can be dis-
cerned that little unbound ligand remained in solution. Hexa-
nucleotide-bridged 13 formed a binding motif that bound
cAMP and FAD with approx. 20 µM Kd, but both ATP and
cGMP too poorly to give a reliable dissociation constant
(Table 3). The shorter tetranucleotide loop of 14 led to
Fig. 6 (a) UV-melting curves of triplex 17 : 19 alone and in the presence
of cAMP, FAD, or ATP. Conditions: 1 µM DNA, 5 µM ligand, 10 mM phos-
phate buffer (pH = 6), 1 M NaCl, heating rate 1 °C min−1. (b) UV/VIS-
spectra of filtrate of triplex 17 : 19 with cAMP. Conditions: 10 µM DNA,
10 µM cAMP, 10 mM phosphate buffer (pH = 6), 1 M NaCl; centrifugation increased affinity for all complementary ligands, including
at 4 °C, using filters with a cut-off of 3 kDa.
ATP, but no detectable binding of cGMP. A hexaethylene glycol
bridge (15) improved binding of ATP, but had a modest effect
on the stability of complexes with cAMP and FAD. As with all
other triplexes, no binding of cGMP was detected. Nor did the
control triplex 18 : 19 show affinity of any of the nucleotides
tested.
Table 2 UV-melting points (Tm’s) of triplexes of 13–18 and 19 in the
absence or in the presence of ATP, cAMP, or FADa,b
Very tight binding of cAMP was found for both 16 : 19 and
17 : 19. Here, dissociation constants below 50 nM were deter-
mined, which is close to the detection limit of our absorption-
based assay. The strongest complex was found for cAMP and
17 : 19, with a Kd of 30 nM. Tight binding was confirmed inde-
pendently by analyzing the complex retained by the filter via
HPLC (Fig. S25, ESI†). For both ATP and FAD, the triplex with
the propylene chain strand gave the lowest Kd value, though,
suggesting that it is the better binding motif for these larger
ligands.
Sequences Bridge DNA only ATP
cAMP
36.9
FAD
37.2
13 : 19
14 : 19
15 : 19
16 : 19
17 : 19
18 : 19
T6
T4
HEG
C3
AS
A
37.3
37.1
34.0/44.0c 34.1/44.1c 34.6/44.4c 35.2/44.2c
38.4
40.0
40.7
60.6
38.9
40.6
41.9
60.3
39.7
44.4
45.4
60.3
38.5
41.1
41.9
60.9
a Conditions: 1 µM DNA, 5 µM ligand, 10 mM phosphate buffer
(pH = 6), 1 M NaCl, heating rate 1 °C min−1 b A single UV-transition,
encompassing the triplex-to-duplex and duplex-to-single-strand
transitions were observed. c Separate triplex-to-duplex and duplex-to-
single-strand transitions observed, both melting points given.
.
Discussion
effect, with a ΔTm of 4.4 °C in the triplex-to-duplex transition.
The addition of Mg2+ (10 mM) resulted in a slight increase in
the UV-melting point (+1.3 °C). When the pH was raised to a
value of 7.0, melting occurred 6.0–7.5 °C lower than at pH 6.0,
where triplexes containing cytosines are more stable (Table S1,
ESI†). Interestingly, the drop was slightly less pronounced in
the presence of cAMP as ligand.
Several aspects of the current results are interesting. The fact
that neither of the triplexes bound guanine-containing cGMP
and that control triplex 18 : 19 lacking a binding site did not
show affinity for any of the ligands tested suggests that
binding does indeed occur by a combination of Watson–Crick
and Hoogsteen binding. Further, preorganization appears to
be advantageous for tight binding, both on the side of the
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triplex motif and on the side of the ligand, as suggested by the
Further, one may ask whether anything can be learned
low dissociation constant measured for the complex of 17 : 19 from the stability of the triplex motifs prior to ligand binding
and cAMP. In this triple helical motif, the entire structure but (and thus the level of preorganization) and the stability of the
the nucleobase itself was retained from a continuous triple desired complexes with our ligands. The UV-melting points
helix. The cyclic AMP is also significantly more rigid than listed in Table 2 allow a measure of how well the triplex-
adenosine monophosphate, as the annelation freezes out the forming strand or segment is anchored on the underlying
conformational flexibility of the ring.
duplex regions. “Carving out” the binding site destabilizes the
Earlier studies of DNA and RNA triplexes binding cGMP triplex thermally, with the single melting transitions shifted to
had led to a hexanucleotide loop as preferred linker.25 Our lower temperatures by at least 20 °C for all binding motifs,
structure search now led to much shorter linkers, with a back- compared to control triplex 18 : 19 (third column of Table 2).
bone close to that of a single nucleotide residue but lacking Starting with the constructs featuring oligonucleotide
the adenine base (16 and 17) as the preferred binders, with the loops (13 and 14), melting points then gradually increase, with
highest affinity for the ligands tested. The HEG bridge of 15 HEG-linked triplex 15 : 19 melting slightly higher and those
has a length between that of the hexa- and tetranucleotide featuring the C3 or abasic site moiety giving melting
loops of 13 and 14, and the short linkers that were modeled points that are higher still (16 : 19 and 17 : 19). A plot of the
after a single nucleotide.
Naïvely, the observation that bridges with the backbone complexes with the three ligands tested (Fig. 8) shows a
length of a natural nucleotide (C3, AS) should allow the rough correlation. close numerical correlation would
triplex melting points against the dissociation constants of
A
binding of an adenine phosphate ligands at all, is surprising, have been unreasonable to expect, as this level of analysis
as the backbone of the triplex and the ribose phosphate ignores alternative binding modes (Fig. 7) and thus possible
should get into a steric conflict (Fig. 7, binding mode A). conformations of the triplex that may be stable but are
However, a more careful inspection of possible binding modes unproductive.
reveals that there is a second, alternative binding orientation,
Some unproductive conformations that may exist are shown
with the nucleobase flipped by 180° (binding mode B in in cartoon format in Fig. 9. In either design (terminal binding
Fig. 7).49 The abasic site-bridged triplex has a more rigid, site or interior binding site), a blocked state can readily be
cyclic linker structure, and can be expected to be slightly better imagined. For clamp-containing structures, the stacking
preorganized towards ligand binding than the C3-containing moiety may stack directly on the terminal base triplet (state V),
analogue with its acyclic backbone. However the abasic site- thus preventing the ligand from binding. In bridged systems, a
containing triplex will almost certainly suffer the steric clash well-stacking or well preorganizing linker can be expected to
that prevents binding mode A, resulting in an entropically less favor the productive, open state (VII), making the blocked
favorable situation. This may explain why 16 produces the state, with the complementary thymidines bulged out (VIII),
better binders for two of the three adenosyl ligands tested, as less likely, and thus preorganizing the binding motif toward a
compared to 17. It is unclear whether the propylene chain of complex with the ligands. Unless in doing so the linker
the C3-bridge fully suppresses binding mode A, or allows it, blocks one binding mode of the adenine base (Fig. 7), it will
with slightly distorted backbone geometry in the linker most probably favor strong binding, both on enthalpic and
between the oligopurine segments. We are currently initiating entropic grounds. This may, at least in part, explain why abasic
a project aimed at elucidating the full three-dimensional struc- site-bridged triplex 17 : 19 forms such a stable complex with
ture of C3-bridged triplex with nucleotide ligands bound in cAMP.
order to shed light on this.
Fig. 7 Two orientations allow an adenosyl ligand to bind via a combi-
nation of Watson–Crick and Hoogsteen base pairing. Binding mode A
differs from binding mode B by a 180° flip of the ligand and avoids a
steric clash of the ribose phosphate (abbreviated as a ball) and the linker
backbone of the oligopurine strand (shown as a wobbly line).
Fig. 8 Correlation between triplex melting temperature in the absence
of a ligand and dissociation constants of complexes formed with ATP,
cAMP or FAD as ligands. See Tables 2 and 3 for details.
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important factor in the binding of adenosine phosphates by
triplex-based DNA binding motifs that can engage in Watson–
Crick and Hoogsteen base pairing.
Experimental
General
Ligands cAMP, SAM, and ATP as well as the reagents were from
Sigma-Aldrich/Fluka (Deisenhofen, Germany), Acros (Geel,
Belgium), or Fluorochem (Karlsruhe, Germany). Unmodified
oligonucleotides and strand 15–17 were used obtained from
Biomers (Ulm, Germany) in HPLC-purified form and were
without further purification. Flavin adenine dinucleotide was
obtained from Alfa Aesar (Karlsruhe, Germany), cGMP was
from Carbosynth (Compton, Berkshire, UK). The reagents
2-cyanoethyl N,N-diisopropylchlorophosphoramidite and 17-O-
(4,4′-dimethoxytrityl)-hexaethyleneglycol-1-[(2-cyanoethyl)-(N,N-
diisopropyl)]-phosphor-amidite were from ChemGenes
(Wilmington, USA). All chemicals used for automated solid-
phase DNA synthesis were from Proligo (Hamburg, Deutsch-
land), including the unmodified phosphoramidites of deoxy-
nucleosides, DCI activator (0.25 M 4,5-dicyanoimidazol in
acetonitrile), the cpg loaded with the first deoxynucleoside
(pore size 1000 Å, 27–32 µmol g−1 loading), amidite diluent
(abs. acetonitrile, ≤90 ppm water), tetrazole solution (0.45 M
in abs. acetonitrile), cap B (THF/pyridine/1-methylimidazole
80 : 10 : 10 v/v/v), cap A (THF/tert-butylphenoxyacetic anhydride
100 : 5 v/v), oxidizer solution (0.1 M iodine in THF/pyridine/
water 77 : 21 : 2 v/v/v), and deblock solution (trichloroacetic
acid/dichloromethane 3 : 97 v/v).
N-2-[(2-O-4,4′-Dimethoxytritylethoxy)ethyl]-N′-2-(2-hydroxy-
ethoxy)ethyl-3,4,9,10-perylenetetracarboxylic diimide (20). The
protocol is a modification of the method of Rahe et al.40 Pery-
lenetetracarboxylic diimide (1.16 g, 3.0 mmol, 1.5 eq.) was
added to a slurry of zinc acetate dehydrate (1.16 g, 3.0 mmol,
1.5 eq.) in dry pyridine (10 mL), followed by the addition of a
solution of 2-(2-aminoethoxy)ethanol (0.41 g, 4.0 mmol, 2 eq.)
and 2-(2-O-4,4′-dimethoxytritylethoxy)ethylamine (0.80 g,
2.0 mmol, 1 eq.) in dry pyridine (20 mL). The reaction mixture
was heated to 125 °C under a nitrogen atmosphere for 16 h.
The solvent was removed under reduced pressure. Then, the
residue was dissolved in dichloromethane (15 mL) and the
solution filtered. The filtrate was evaporated to dryness and
the crude product purified by column chromatography
(dichloromethane/methanol/trimethylamine, 98 : 1 : 1, v/v/v) to
yield 0.41 g (4.7 mmol, 25%) of compound 20 as a red solid.
TLC, dichloromethane/methanol/trimethylamine, 98 : 1 : 1,
v/v/v: Rf = 0.28. The spectroscopic data were in agreement with
the data reported in literature.39
Fig. 9 Possible equilibria between triplex motifs with productive,
accessible binding sites (states IV, VII), blocked binding sites (V, VIII) and
complexes with ligand A (VI, IX).
Conclusion
Among the two different designs of triplex-based DNA binding
motifs tested, the one with a lock bridging the oligopurine
strands of two triplex segments was found to bind adenosine-
containing ligands more tightly than the one with a binding
site at the terminus of a single triple helix. Among the ligands
tested, cAMP gave the lowest dissociation constants, lower
than those of the larger ligands with much more total stacking
surface (FAD) or electrostatically more favorable ligands (SAM).
Further, the tightest complex was found for a triplex with a
short bridge with a sterically demanding and conformationally
restricted abasic nucleotide analog (17 : 19 : cAMP, Kd
=
N-2-[(2-O-4,4′-Dimethoxytritylethoxy)ethyl]-N′-2-{[2-O-(2-cyano-
ethyldiisopropylchlorophosphino)ethoxy]ethyl}-3,4,9,10-peryl-
30 nM). This level of affinity rivals that of riboswitches binding
nucleobases by engulfing them in the interior of their folded
structure.50 In our case, thermal stability of ligand-free
triplexes correlates roughly with binding affinity towards ATP,
FAD and cAMP. These results point to preorganization as an
entetracarboxylic diimide (10). The following is
a slight
modification of the synthesis described by Rahe et al.40
Compound 20 (50 mg, 0.06 mmol, 1 eq.) was dried for 16 h at
0.2 mbar and dissolved in N,N-diisopropylethylamine
5740 | Org. Biomol. Chem., 2015, 13, 5734–5742
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Paper
(0.05 mL, 0.29 mmol, 5 eq.) and dry dichloromethane solutions (510 µL) were heated to 55 °C and allowed to cool to
(0.15 mL) under argon atmosphere. Then, 2-cyanoethyl N,N- r.t. in 3 h. After 30 min at r.t., samples were cooled to 4 °C and
diisopropylchlorophosphitamidite (30 µL, 0.12 mmol, 2 eq.) kept at this temperature overnight. The solutions were then
was added, and the solution was stirred for 2 h at r.t. The loaded on Amicon ultracentrifugal filter units from Millipore
product was precipitated with petroleum ether and the remain- with a molecular weight cutoff of 3 kDa and centrifuged for
ing solvent removed after centrifugation to yield 27 mg 1 h at 14 000g and 4 °C. The filtrates (450 µL) were diluted with
(0.03 mmol, 42%) of product as a red solid that was used water (150 µL), and the absorption was measured in the range
in the subsequent reaction without further purification. The of 600–220 nm. The concentration of ligand in the filtrate
spectroscopic data were in agreement with those in the (unbound ligand) was calculated according to Lambert-Beer’s
literature.39
law with the following extinction coefficients: ε260(cAMP) =
12 300 M−1 cm−1, ε260(ATP) = 15 400 M−1 cm−1, ε260(SAM) =
15 400 M−1 cm−1, ε450(FAD) = 11 300 M−1 cm−1, ε254(cGMP) =
Synthesis of modified DNA strands 2–6
Modified oligonucleotides were synthesized on a 8909 Expe- 13 600 M−1 cm−1
dite synthesizer (Perseptive Biosystems Foster City, CA) with
.
UV melting curves
Expedite Workstation as software (version 2.5) according to the
1 µmol standard protocol. For modified phosphoramidites
(8–10) 0.1 M solutions in acetonitrile were used, and the coup-
ling times were extended to 440 s. All strands were deprotected
and cleaved from solid support by treatment with aqueous
ammonia at 55 °C for 16 h. Crudes were purified by reversed-
phase HPLC on a Dynamax System (Rainin) with an EC 250/4.6
Nucleosil 120-5 C4 (Macherey-Nagel) column and a gradient of
acetonitrile in 0.1 M triethylammonium acetate buffer, pH 7.0.
Yields were determined by UV-absorption at 260 nm.
The UV melting curves were measured on a Lamda 25 spectro-
photometer (Perkin Elmer) with peltier-based temperature
control. Sample were 1 µM oligonucleotides in 10 mM phos-
phate buffer, 1 M NaCl, pH = 6.0 with or without 5 µM ligand.
Absorption was measured at 260 nm in the temperature range
between 5 °C and 85 °C with a heating and cooling rate of 1 °C
min−1. For each sample, two heating and cooling curves were
recorded. The melting points were calculated as the extrema of
the first derivation of the heating curves using UV-WinLab
(Perkin Elmer).
3′-TTCTCTT-(HEG)-TTCTCTT-5′ (2). HPLC, 0% acetonitrile
for 5 min, then gradient of 0% to 10% CH3CN in 20 min, 10%
to 20% in 25 min, 20% to 25% in 10 min, then 25% to 80% in MALDI-TOF mass spectrometry
5 min, tR = 36 min. Yield 23%. MALDI-TOF MS, calculated for
The MALDI-TOF mass spectra were measured on a Bruker
Reflex IV or Microflex spectrometer in linear negative mode
with an external calibration. For sample preparation 0.5 µL of
the oligonucleotide solution (10−6–10−8 M in water) were put
on an MTP AnchorChip™ target (Bruker) and evaporated to
dryness at 5 mbar. Then, 0.5 µL of a matrix/comatrix solution
(0.3 M 2,4,6-trihydroxyacetophenone monohydrate in ethanol/
0.1 M diammonium citrate in water; 2 : 1 v/v) were pipetted
onto the spot and allowed to dry at ambient pressure. The
target was then introduced into the mass spectrometer.
C
148H204N32O101P14 [M − H]− 4478 Da, found 4476.
3′-TTCTCTTTTT-(Py)-TTCTCTT-5′ (3). HPLC, 0% acetonitrile
for 5 min, then gradient of 0% to 10% CH3CN in 20 min, 10%
to 20% in 25 min, 20% to 25% in 10 min, then 25% to 80% in
5 min, tR = 42 min. Yield 6%. MALDI-TOF MS, calculated for
C
187H235N38O118P17 [M − H]− 5426 Da, found 5427.
3′-TTCTCTT-(PDI)-TTCTCTT-5′ (4). HPLC, gradient of 0% to
12% acetonitrile in 15 min then 12% to 20% in 40 min, tR
=
38 min. Yield 3%. MALDI-TOF MS, calculated for
C
168H204N34O102P14 [M − H]− 4763 Da, found 4764.
3′-TCCTCTT-(PDI)-TTCTCCT-5′ (5). HPLC, 0% acetonitrile
for 5 min, then gradient of 0% to 10% CH3CN in 20 min, 10%
to 20% in 25 min, 20% to 25% in 10 min, then 25% to 80% in
5 min, tR = 46 min. Yield 16%. MALDI-TOF MS, calculated for
Acknowledgements
This work was supported by Deutsche Forschungsge-
meinschaft (DFG, grant no. RI 1063/13-1 to C.R.). The authors
thank A. Göckel for discussions, and H. Griesser, Dr E. Kervio
and M. Kramer for a review of the manuscript.
C
150H178N30O88P12 [M − H]− 4734 Da, found: 4735.
3′-TCCTCTT-(PDI)-TTCTCCTTTTTAGGAGA-5′
(6). HPLC,
column temperature 55 °C, 0% acetonitrile for 5 min, then gra-
dient of 0% to 10% CH3CN in 20 min, 10% to 20% in 25 min,
20% to 25% in 10 min, then 25% to 80% in 5 min, tR
=
42 min. Yield 5%. MALDI-TOF MS, calculated for
C
Notes and references
266H326N72O161P25 [M − H]− 7875 Da, found: 7881.
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