8-vinyl-2′-deoxyguanosine as a fluorescent 2′-deoxyguanosine mimic for investigating DNA hybridization and topology

Article abstract of DOI:10.1002/anie.201100078

A one light strand: 2′-Deoxyguanosine was made fluorescent by attaching a vinyl group at the C8 position (see structure). The resulting fluorophore is highly sensitive towards DNA double-strand formation and alterations in the secondary structure of its p

Full text of DOI:10.1002/anie.201100078

Communications
DOI: 10.1002/anie.201100078
Fluorescent Guanine
8-Vinyl-2’-deoxyguanosine as a Fluorescent 2’-Deoxyguanosine Mimic
for Investigating DNA Hybridization and Topology**
Andrꢀ Nadler, Julian Strohmeier, and Ulf Diederichsen*
Fluorescent analogues of the natural nucleobases are widely
used as molecular probes, for example, for investigating
oligonucleotide conformation, detecting DNA hybridization,
or single nucleotide polymorphisms.[1] Typical approaches are
fluorophore-base conjugates[2] in which common fluoro-
phores are tethered to the nucleobases, or expanded nucle-
obases[3] or nucleotides bearing a fluorescent aromatic moiety
instead of a natural nucleobase.[4] Molecular probes for
investigating oligonucleotide conformation and dynamics,
however, should perfectly mimic the natural nucleobases to
minimize structural disturbances and thus resemble the
natural nucleobases with respect to their molecular shape
and hydrogen-bonding pattern.[5] The earliest known and
extensively used adenine analogue 2-aminopurine (AP) is the
most prominent example of this class of molecules.[6] AP was
used in a number of applications including investigation of
base stacking,[7] extruded nucleobases,[8] metal-ion coordina-
tion,[9] and protein-induced conformational changes of DNA
secondary structure.[10] Other fluorescent nucleobase ana-
logues closely resemble their parent nucleobases, mostly
pyrimidine and adenine mimics which include the title
compound 8-vinyl-adenine, an adenine analogue.[11] There
are few examples for guanine analogues that match the
requirements for a suitable fluorescent probe, mainly iso-
xanthopterins, 8-azaguanine which exhibits anion fluores-
cence at higher pH values, 8-furyl-guanine which has yet to be
incorporated into oligonucleotides, and very recently 8-(2-
pyridinyl)-guanine.[11d,12] Our approach is directed towards
the synthesis of a guanine analogue that maintains the
nucleobase core while being made fluorescent in its neutral
form by minimal substitution. Furthermore, the fluorescent
nucleoside should be able to adopt both the nucleoside syn
and anti conformation, which is a prerequisite for the
formation of various G-quadruplex structures and the left-
handed Z-DNA helix.
requirements while its emission properties are very sensitive
towards changes within its microenvironment, such as the
formation of different quadruplex structures. 2’-Deoxygua-
nosine was used as a starting material in the synthesis of the
VdG phosphoramidite building block 2. Bromination at the
C8 position and subsequent protection of the exocyclic amino
group and the hydroxy groups yielded compound
3
(Scheme 1).[13] Nucleoside 3 was subjected to Stille coupling
Scheme 1. Synthesis of the VdG phosphoramidite building block 2,
nucleoside 1 and nucleobase 7. R=TBDMS. DIPEA=N,N-diisopropy-
lethylamine, DMT=dimethoxytrityl, TBDMS=tert-butyldimethylsilyl,
TBAF=tetrabutylammoniumfluoride, TFA=trifluoracetic acid.
The newly introduced fluorescent 2’-deoxyguanosine
mimic 8-vinyl-2’-deoxyguanosine (VdG, 1) matches these
with tributylvinyltin to give the vinyl-substituted 2’-deoxy-
guanosine derivative 4 in good yields. TBAF deprotection of
the TBDMS groups yielded nucleoside 5 which was converted
into the 5’-DMT protected compound 6.[14] The VdG phos-
phoramidite building block 2 was generated using an estab-
lished method.[15] In addition, nucleoside 1 and the respective
nucleobase 7 were prepared from the protected nucleoside 5.
An extinction coefficient of (7.2 Æ 0.03) LmmolÀ1 cmÀ1 at
260 nm was determined for VdG, which because of a red-
shifted absorption maximum, is distinctly lower than the
[*] Dr. A. Nadler,[+] J. Strohmeier,[+] Prof. Dr. U. Diederichsen
Institut fꢀr Organische und Biomolekulare Chemie
Georg-August-Universitꢁt Gꢂttingen
Tammannstrasse 2, 37077 Gꢂttingen (Germany)
Fax: (+49)551-39-22944
E-mail: udieder@gwdg.de
[+] These authors contributed equally to this work.
[**] We thank Heinrich Prinzhorn for measuring ESI-MS spectra of the
telomeric DNA sequences. Generous financial support from the
DFG (IRTG 1422) is gratefully acknowledged.
corresponding
value
of
2’-deoxyguanosine
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201100078.
(12.18 LmmolÀ1 cmÀ1)[16]. VdG exhibits the highest emission
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when irradiated at 277 nm but excitation at wavelengths up to
325 nm is also feasible allowing for site-specific probing of
larger oligonucleotides. The quantum yield of VdG was
determined relative to the respective AP nucleoside and
found to be 0.72 Æ 0.03 (see Supporting Information). The
fluorescence properties of nucleoside 1 were examined at
different pH values. The fluorescence intensity appeared to
be unaffected by changes of pH value within the physiological
relevant region between pH 5–9. The fluorescence intensity at
lower and higher pH values is affected by protonation or
deprotonation of the vinylguanine moiety (Supporting Infor-
mation). A partial depurination was observed upon pro-
longed exposure to acidic solvents (3 days at pH 3).
mined also after formation of B-DNA double strands by
addition of the respective complementary strands 5’-
d(CTAXCXATC) (X = dG (O5), dA (O6), dT (O7), dC
(O8)).
Quenching of VdG fluorescence in dsDNA turned out to
be comparable but more pronounced than single-strand
results. Neighboring guanine and thymine nucleobases led
to highest quenching of VdG fluorescence whereas adjacent
cytosine and especially adenine were less influential
(Figure 2).
The base pairing of VdG incorporated in double stranded
DNA (dsDNA) was investigated by temperature dependent
UV spectroscopy. The duplex stability of 5’-d(GATCGC-
TAG) + 5’-d(CTAGCGATC) (melting temperature Tm =
328C) was compared to the stabilities of an oligomer that
contains VdG in the central position 5’-d(GATCVdGCTAG)
(O1) together with the complementary strands 5’-d(CTAGX-
GATC) (X = dC, dT, dG, dA, abasic site) varying with respect
to the opposed nucleobase position (Figure 1). The double
strand with VdG–dC base pair turned out to be only 48C less
stable, whereas mismatch pairing with dG or dT led to a drop
of stability of about 148C. Stable duplexes were not even
detected for dA and abasic site mismatches.
Figure 2. Emission spectra of duplexes O1+O5, O2+O6, O3+O7,
O4+O8 (duplex concentration: 500 nm, 58C, 10 mm phosphate buffer
pH 7, 100 mm NaCl, excitation wavelength: 277 nm).
DNA double-strand formation can be monitored by VdG
incorporation as indicated by decreasing emission upon
formation of B-form helices. This was indicated by comparing
the fluorescence spectra of the oligomers O1 + O5, O2 + O6,
O3 + O7, O4 + O8, detected as single-stranded oligomers at
temperatures above the melting temperature (408C/508C)
and duplexes at 58C. The intensity of VdG emission was
found to be virtually identical with samples containing only
the single-stranded oligomers O1, O2, O3, and O4 at elevated
temperatures whereas significant fluorescence quenching was
observed at temperatures below the respective melting
temperatures of the duplexes (Figure 3a,b and Supporting
Information).
Duplex formation with B-DNA topology was confirmed
by comparison of circular dichroism (CD) spectra and UV
melting curves of VdG functionalized duplexes (O1 + O5,
O2 + O6, O3 + O7, O4 + O8) with the respective guanine-
containing duplexes O9 + O5, O10 + O6, O11 + O7, O12 +
O8 (5’-d(GATXGXTAG), X = dC (O9), dT (O10), dA (O11),
dG (O12)). VdG incorporation into dsDNA appeared to have
only negligible effects on the formation of a B-form DNA
helix and on the double-strand stabilities (DTm = 1–58C)
(Figure 3c,d and Supporting Information).
In a second application, the VdG fluorophore was used as
sensor for different DNA G-quadruplex topologies. VdG was
incorporated into human telomeric DNA 5’-d[AGGG-
(TTAGGG)3T] (O13). In the presence of its complementary
strand a regular B-DNA duplex is formed, whereas the single-
stranded telomeric DNA folds into quadruplex structures
with topologies that depend on temperature, pH value, and
the respective metal ion.[17] In aqueous NaCl solution a
quadruplex structure is predominant with two edgewise and
one diagonal connecting TTA loops between the respective
Figure 1. UV melting curves of duplexes formed between
5’-d(GATCVdGCTAG) (O1) and the respective complementary strand
5’-d(CTAGXGATC) (X=dC, dA, dG, dT, abasic site) and for comparison
the natural dsDNA 5’-d(GATCGCTAG) + 5’-d(CTAGCGATC) (2.5 mm,
10 mm phosphate buffer pH 7, 100 mm NaCl, 260 nm).
Fluorescence of VdG is likely to depend on the neighbor-
ing nucleobases. Therefore, a series of oligonucleotides with
the general sequence 5’-d(GATXVdGXTAG) (X = dC (O1),
dT (O2), dA (O3), dG (O4)) was investigated. A pronounced
9–12-fold reduction of emission intensity was observed upon
incorporating VdG (1) into single-stranded DNA (ssDNA)
oligonucleotides that do not adopt distinct conformations
(Supporting Information). VdG fluorescence was quenched
most effectively by neighboring guanine or thymine bases
whereas adjacent cytosine and adenine moieties led to higher
residual emission (Supporting Information). Since quenching
caused by neighboring bases can be monitored most mean-
ingful within a uniform topology, fluorescence was deter-
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Figure 3. Fluorescence quenching upon double-helix formation, for O2 and O2+O6. Emission spectra of O2 and O2+O6 at a) 58C. b) 408C.
(500 nm, 10 mm phosphate buffer pH 7, 100 mm NaCl, excitation wavelength: 277 nm). c) Melting curves of VdG modified duplex O2+O6 and
guanine duplex O10+O6 (2.5 mm, 10 mm phosphate buffer pH 7, 100 mm NaCl, 260 nm). d) CD spectra of VdG modified duplex O2+O6 and
guanine duplex O10+O6 (5 mm, 10 mm phosphate buffer pH 7, 100 mm NaCl).
triple-G repeats (Figure 4).[17a–c] In aqueous KCl solution,
however, the predominant quadruplex forms are character-
ized by a double chain reversal connecting TTA loop
spanning one flank of the quadruplex core (Figure 4b).[17c]
To examine VdG as a fluorescence sensor with different roles
in the quadruplex (central/terminal position and syn/anti
conformation) the telomeric sequence 5’-d[AGGG-
(TTAGGG)3T] (O13) was modified with the VdG nucleotide
at positions G3, G4, or G15, respectively. The emission
properties of the VdG-functionalized oligonucleotides
unmodified O13 in NaCl and KCl solution. For duplex
formation with O17 the CD spectra were found to be almost
identical (Figure 5, and Supporting Information). The CD
spectra of all single-stranded oligomers O13–O16 clearly
indicated the formation of NaCl and KCl-forms irrespective
of the VdG modification and were in agreement with
published data (Figure 5, and Supporting Information).[19]
Only slight deviations with respect to the intensity of the
respective Cotton effects were observed in some cases when
comparing the spectra of O13 with the CD data of O14, O15,
and O16 (Figure 5, and Supporting Information). Three of the
quadruplex structures require the VdG nucleotide in syn and
three in anti conformation indicating that the VdG nucleotide
can be incorporated almost equally well as a mimic for anti-
dG and for syn-dG.
The fluorescence properties of the VdG modified oligo-
mers O14–O16 were closely correlated to the respective
topology. The emission spectra of the duplexes O14–O16 +
O17 exhibited emission maxima at 398 nm (Figure 6, and
Supporting Information) as it was observed in the case of
duplex formation of oligomers O1–O4 (Figure 2); no salt
dependency was indicated.
5’-d[AGVdGG(TTAGGG)3T]
(TTAGGG)3T] (O15),
(O14),
and
5’-d[AGGVdG-
5’-d[A-
(GGGTTA)2GVdGGTTAGGGT] (O16) were investigated
in aqueous KCl and NaCl solution.
The influence of VdG incorporation on the thermal
stability of the respective quadruplex folds and the B-type
duplexes formed by oligomers O14–O16 with their comple-
mentary strand d[A(CCCTAA)4T] (O17) was investigated by
means of temperature-dependent UV spectroscopy. A slight
rise in the thermal stability of the duplex (DTm = + (1–2)8C)
and quadruplex structures (DTm = + (0–5)8C) was observed
(see Supporting Information). Therefore, VdG incorporation
does not significantly affect the stability of telomeric DNA
topologies.
The emission maxima of the quadruplex forming oligo-
mers O14–O16 were blue shifted by approximately 4–8 nm
with respect to the double-stranded oligomers (Figure 6, and
Supporting Information). It can clearly be differentiated,
Furthermore, CD spectra of the respective oligomers
O14–O16 were compared to the spectra obtained from
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Angew. Chem. Int. Ed. 2011, 50, 5392 –5396

Figure 6. Emission spectra of single-stranded VdG modified human
telomeric oligonucleotide O14 and the duplex O14+O17 in the
presence of NaCl and KCl (500 nm, 10 mm phosphate buffer pH 7,
135 mm NaCl or 100 mm KCl, excitation wavelength: 277 nm).
a central G-tetrad layer is required, because the emission
spectra of both quadruplex forms of oligomer O15 with VdG
in the terminal layer were indistinguishable. Nevertheless,
quadruplex formation was still detectable for oligomer O15
by a twofold higher emission intensity compared to the
respective duplex. Overall, VdG can be incorporated in
different kinds of DNA topologies. Fluorescence readout of
VdG was found to be indicative for monomeric, double
helical and quadruplex secondary structures, quadruplex
topology, and position of the G-tetrad layer.
8-Vinyl-2’-deoxyguanosine (VdG) was developed as a
novel fluorescent 2’-deoxyguanosine analogue, which resem-
bles the guanine base pairing in DNA double strands and
quadruplex structures with respect to hydrogen bonding and
geometrical requirements. It thus offers a robust and sensitive
fluorescence sensor for microenvironment detection. The
nucleotide VdG is considered to have a potential comparable
to 2-aminopurine that is widely used as fluorescent adenine
analogue. We have shown a convenient synthesis of the VdG
phosphoramidite building block for solid-phase DNA syn-
thesis and the use of VdG as a fluorescence probe for DNA
base pairing and formation of different topologies. A
correlation of the fluorescence properties of VdG with the
monomer–duplex transition, the type of neighboring nucle-
obase, and the respective conformation of the oligomer was
found for VdG-containing double strands. VdG was further
used to differentiate between topologies of G-quadruplexes.
VdG was shown to be capable of adopting both the nucleotide
syn and anti conformation required for distinct quadruplex
structures. Thus VdG provides a sensitive fluorescent tool
within nucleic acid chemistry.
Figure 4. Schematic representation of quadruplex solution structures
adopted by human telomeric DNA a) NaCl form. b) KCl form.[18]
Syn dG residues are light gray, anti dG residues are dark gray, arrows
indicate the strand orientation (5’ to 3’). The respective positions of
the VdG substitution are indicated with capital letters A (O14),
B (O15), C (O16).
Received: January 5, 2011
Published online: May 3, 2011
Figure 5. CD spectra of single-stranded human telomeric DNA oligo-
nucleotides O13 and O14 and the respective duplexes with the
complementary strand O17 under a) NaCl conditions (4 mm, 58C in
10 mm phosphate buffer pH 7, 135 mm NaCl) and b) KCl conditions
(4 mm, 58C, 10 mm phosphate buffer pH 7, 100 mm KCl).
Keywords: DNA structures · fluorescent probes · G-tetrads ·
.
nucleobases · oligonucleotides
whether VdG-labeled DNA oligomers form monomers,
dimers, or a tetramer fold.
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