Synthesis and hybridization properties of oligonucleotides modified with 5-(1-aryl-1,2,3-triazol-4-yl)-2′-deoxyuridines

Article abstract of DOI:10.1039/c2ob26717a

Oligonucleotides modified with consecutive incorporations of 5-(1-aryl-1,2,3-triazol-4-yl)-2′-deoxyuridine monomers X-Z display strong thermal affinity and binding specificity toward RNA targets, due to formation of chromophore arrays in the major groove.

Full text of DOI:10.1039/c2ob26717a

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COMMUNICATION
Synthesis and hybridization properties of oligonucleotides modified with
5-(1-aryl-1,2,3-triazol-4-yl)-2′-deoxyuridines†‡
Mamta Kaura, Pawan Kumar and Patrick J. Hrdlicka*
Received 30th August 2012, Accepted 24th September 2012
DOI: 10.1039/c2ob26717a
studied in detail,21,23 the question how the size of the aromatic
Oligonucleotides modified with consecutive incorporations of
moiety influences stacking efficiency and thermostability has not
been systematically addressed. Following a hypothesis that
chromophores with larger aromatic surfaces are likely to result in
stronger stacking interactions in the spacious major groove, we
set out to study the hybridization properties of ONs, which are
5-(1-aryl-1,2,3-triazol-4-yl)-2′-deoxyuridine monomers X–Z
display strong thermal affinity and binding specificity toward
RNA targets, due to formation of chromophore arrays in the
major groove.
The use of nucleic acids as scaffolds for organization of chromo-
phore arrays is an area of considerable focus, which is fuelled by
the promise for materials with interesting photophysical and
electronic properties.1–6 A frequently employed approach toward
this end entails self-assembly of duplexes involving oligonucleo-
tides (ONs), which are densely modified with chromophore-
functionalized nucleotide monomers. Examples of building
blocks include monomers where chromophores replace nucleo-
base moieties7–9 or are attached to non-nucleosidic linkers,10,11
sugar skeletons12–14 or nucleobase moieties. Among the latter
class, C5-functionalized pyrimidine monomers in which chromo-
phores are either directly attached to the nucleobase moiety or
attached via an alkynyl linker have been studied in particular
detail and demonstrated to facilitate array formation in the major
groove.15–19 While array formation often partially counteracts
the prominent duplex destabilization induced by these mono-
mers, the resulting duplexes still only display moderate thermo-
stability. Development of nucleotide building blocks, which
enable formation of chromophore arrays in the major groove
without compromising duplex thermostability, therefore remains
a desirable goal.
modified
with
5-(1-aryl-1,2,3-triazol-4-yl)-2′-deoxyuridine
monomers X–Z featuring three differentially sized aromatic
moieties at the 1-position of the triazole ring (Fig. 1).
Phosphoramidites 3X and 3Y were obtained via the same
general strategy, which we recently used for the synthesis of 3Z
(Scheme 1).24 Thus, O5′-protected 5-ethynyl-2′-deoxyuridine
125 was reacted with azidobenzene26 or 1-azidonaphthalene27 in
a CuI catalyzed [3 + 2] azide–alkyne cycloaddition28 to afford
nucleosides 2X and 2Y in 74% and 78% yield. Subsequent O3′-
phosphitylation using 2-cyanoethyl-N,N′-diisopropylchlorophos-
phoramidite (i.e., PCl-reagent) and N,N′-diisopropylethylamine
(DIPEA) provided 5-(1-aryl-1,2,3-triazol-4-yl)-2′-deoxyuridine
phosphoramidites 3X20 and 3Y in 60% and 73% yield,
respectively.
The phosphoramidites were incorporated into ONs via
machine-assisted solid-phase DNA synthesis (hand-coupling
20 min, 4,5-dicyanoimidazole as an activator; coupling yields
>95%, >95% and ∼92% for monomers X, Y and Z, respecti-
vely). The composition and purity of the modified ONs was
verified by MALDI-ToF MS analysis (Table S1‡) and ion-pair
reverse-phase HPLC, respectively.
Nielsen and coworkers have recently demonstrated that ONs,
which are consecutively modified with 5-(1-phenyl-1,2,3-triazol-
4-yl)-2′-deoxyuridine monomers, display strong and highly
specific affinity toward RNA targets due to the formation of
stabilizing chromophore arrays in the major groove.20–22 While
the influence of phenyl substitution on array formation has been
Department of Chemistry, University of Idaho, PO Box 442343,
Moscow, ID 83844-2343, USA. E-mail: Hrdlicka@uidaho.edu;
Tel: +1-208-885-0108
†Dedicated to the memory of Professor H. Gobind Khorana.
‡Electronic supplementary information (ESI) available: General experi-
mental section; synthetic protocols and NMR spectra for nucleosides
2X–3Y; protocols for ON synthesis and purification; MS-data for
modified ONs; description of thermal denaturation experiments; Tm data
recorded in a medium salt buffer; additional fluorescence emission
spectra. See DOI: 10.1039/c2ob26717a
Fig. 1 Structures of 5-(1-aryl-1,2,3-triazol-4-yl)-2′-deoxyuridines
studied herein.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 8575–8578 | 8575

Monomers X–Z were incorporated once, twice or four times
into a 9-mer T-rich sequence that has been used to study and
prepare self-assembling chromophore arrays.20 Thermal denatura-
tion temperatures (Tm’s) of duplexes between modified ONs and
complementary DNA/RNA targets were determined in buffers of
high or medium ionic strength (Tables 1 and S2‡, respectively).
Singly modified ONs display substantially lower thermal
affinity toward DNA and RNA complements than corresponding
unmodified ONs (see ΔTm’s for the B1-series, Table 1), which is
commonly observed for ONs modified with C5-chromophore-
functionalized pyrimidine monomers.15,16,29–31 Duplex thermo-
stability decreases progressively as the size of the aryl substituent
increases, most likely due to increased perturbation of the
hydration spine in the major groove. Incorporation of two 5-(1-
aryl-1,2,3-triazol-4-yl)-2′-deoxyuridine monomers as next-
nearest neighbors results in further duplex destabilization,
although the energetic penalty associated with monomer Y is
partially reversed (see ΔTm/mod for the B2-series, Table 1). In
contrast, incorporation of two or four consecutive X or Y mono-
mers strongly reverses duplex destabilization, especially in X-
modified duplexes with RNA targets (compare ΔTm/mod trends
for X1→X3→X4 and Y1→Y3→Y4, Table 1). Interestingly, Z4,
which features four consecutive incorporations of monomer Z,
displays very high affinity toward RNA as well as DNA targets
(see ΔTm/mod for Z4, Table 1) although it should be noted that
broad transitions are observed (Fig. S4‡).
Computational studies have previously linked the increased ther-
mostability of X4 : RNA to formation of chromophore arrays in the
major groove.21 This, along with the observed Tm trends for Y/Z-
modified duplexes and results from additional structural studies
(vide infra), suggests that incorporation of consecutive 5-(1-aryl-
1,2,3-triazol-4-yl)-2′-deoxyuridine monomers is a general strategy
toward formation of stable chromophore arrays in the major
groove. The inherently destabilizing effect of these building blocks
is, most likely, counteracted by favorable hydrophobic interactions
between chromophores upon array formation. However, the corre-
lation between duplex/array stability and size/hydrophobicity of the
aryl moiety is complex (trend in ΔTm/mod values: Z4 ≥ X4 > Y4).
Next, the thermostability of duplexes between B1- or
B4-series ONs and RNA targets featuring a centrally mis-
matched nucleotide was determined to study the binding specifi-
city of these probes (Table 2). Singly modified ONs display
mismatch discrimination profiles that differ from the correspond-
ing unmodified ONs in the following manner: (i) X1 and Y1
display less efficient discrimination of U-mismatches, (ii) Z1 dis-
plays markedly poorer discrimination of rC-mismatches, and (iii)
Scheme 1 (a) RN3, aq. sodium ascorbate, aq. CuSO4, THF : H2O :
tBuOH, rt (2X: 74%; 2Y: 78%; 2Z:24 52%); (b) PCl-reagent, DIPEA,
CH2Cl2, rt (3X: 60%; 3Y: 73%; 3Z:24 73%); (c) machine-assisted DNA
synthesis. R = phenyl, 1-naphthyl and 1-pyrenyl for the X-, Y- and Z-
series, respectively.
Table 1 Thermal denaturation temperatures (Tm values) for duplexes between B1B4 and complementary DNA/RNA in a high salt buffera
Tm (ΔTm/mod) [°C]
DNA
3′-CACAAAACG
Y
RNA
3′-CACAAAACG
Y
ON
Sequence
5′-GTGT B TTGC
B =
X
Z
X
Z
B1
̲
35.5
[−4.5]
32.0
[−8.0]
25.0b
[−15.0]
35.0
[−3.0]
29.0
[−9.0]
25.0b
[−13.0]
B2
B3
B4
5′-GTG B
̲
̲
̲
TGC
TGC
28.5
[−5.8]
27.5
[−6.3]
—
—
29.5
[−4.3]
29.5
[−4.3]
—
—
5′-GTGT B
32.5
[−3.8]
29.5
[−5.8]
40.0
[+1.0]
32.0
[−3.0]
5′-GTG B
̲
B
̲
GC
38.0
[−0.5]
26.0b
[−3.5]
52.5b
[+3.0]
55.0
[+4.3]
39.0
[+0.3]
55.5b
[+4.4]
a Tm’s determined as the first derivative maximum of thermal denaturation curves (A260 vs. T) recorded in a high salt buffer ([Na+] = 710 mM, [Cl−] =
700 mM, pH 7.0 (adjusted with NaH2PO4/Na2HPO4)), using 1.0 μM of each strand. Tm’s are averages of at least two measurements within 1.0 °C.
ΔTm/mod = change in Tm’s per modification relative to unmodified reference duplexes (+DNA complement: Tm = 40.0 °C; +RNA complement: Tm
=
38.0 °C). “—” denotes no transition. b Weak/broad transition.
8576 | Org. Biomol. Chem., 2012, 10, 8575–8578
This journal is © The Royal Society of Chemistry 2012

Table 2 Tm values for duplexes between the B1/B4-series and
centrally mismatched RNA targetsa
RNA: 3′-CAC AMA ACG
̲
Tm/°C
ΔTm/°C
ON
Sequence
M =
A
C
G
U
D1 5′-GTGTTTTGC
X1 5′-GTGT X TTGC
X4 5′-GTG X GC
Y1 5′-GTGT Y TTGC
Y4 5′-GTG Y GC
Z1 5′-GTGT Z TTGC
Z4 5′-GTG Z GC
38.0
35.0
55.0
29.0
39.0
25.0
55.5
−14.0
−14.5
−20.0
−11.0
−13.5
−6.0
−11.0
−13.5
−7.0
−20.0
−13.0
−20.0
−12.5
−22.0
̲
̲
̲
X
̲
Y
−7.0
−3.0b <−10.0c <−10.0c
Z
−12.5b
−12.5b
−12.5b
a For conditions of thermal denaturation experiments, see Table 1. ΔTm
change in Tm relative to the matched DNA : RNA duplex (M = A).
b Weak transition. c No transition above 15 °C.
=
̲
Fig. 3 Steady-state fluorescence emission spectra of Z4 in the presence
or absence of complementary DNA/RNA or mismatched RNA targets
(λem = 344 nm; T = 10 °C).
(Fig. 3 and S5‡). Interestingly, duplexes between Z4 and cen-
trally mismatched RNA targets display lower excimer intensity
(Fig. 3), which suggests perturbation of the pyrene array as pro-
posed in the structural model (Fig. 2).
In summary, we demonstrate that ONs with stretches of
5-(1-aryl-1,2,3-triazol-4-yl)-2′-deoxyuridine monomers display
improved RNA affinity and specificity relative to reference
strands, due to formation of stabilizing chromophore arrays in
the major groove. This design principle is expected to have
important implications in the design of supramolecular nucleic
acid based π-functional materials and antisense ONs.
Fig. 2 Proposed structural model that rationalizes increased affinity
and specificity of ONs modified with four consecutive 5-(1-aryl-1,2,3-
triazol-4-yl)-2′-deoxyuridine monomers.
X1 and Z1 display improved discrimination of rG-mismatches
(Table 2). Interestingly, the target specificity of X4, Y4 and,
possibly, Z4 is markedly improved relative to their singly
modified counterparts (compare ΔTm for the B4- vs. B1-series,
Table 2). In fact, X4 and Y4 display base pairing fidelity that
compares favorably with the unmodified reference strand,
suggesting that chromophore arrays may have beneficial impacts
on target affinity as well as target specificity. Interestingly, while
ONs with stretches of 5-ethynyl-2′-deoxyuridine monomers are
known to display improved target specificity due to long-range
cooperativity,32 ONs with stretches of C5-chromophore-functio-
nalized 2′-deoxyuridine monomers typically display poor mis-
match discrimination.15,17
The following structural model accounts for the observed Tm-
trends (Fig. 2): (i) hybridization of X4/Y4/Z4 with complemen-
tary RNA targets results in the formation of a stabilizing chro-
mophore array in the major groove, whereas (ii) hybridization
with mismatched targets results in array disruption, reduced
duplex stability and improved mismatch specificity.
Acknowledgements
We appreciate support from Idaho NSF EPSCoR and the
BANTech Center (Univ. Idaho). We thank Drs Lee Deobald and
Sujay P. Sau (both Univ. Idaho) for mass spectrometric analyses.
Notes and references
§Broad peaks in the UV-Vis spectra of Z1–Z4 with/without DNA/RNA
targets precluded determination of hybridization-induced changes in
absorption maxima, which could have provided additional insight into
pyrene binding modes (results not shown).
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