DNA gold nanoparticle conjugates incorporating thiooxonucleosides: 7-Deaza-6-thio-2-deoxyguanosine as gold surface anchor

Article abstract of DOI:10.1021/bc200069j

A new protocol for the covalent attachment of oligonucleotides to gold nanoparticles was developed. Base-modified nucleosides with thiooxo groups were acting as molecular surface anchor. Compared to already existing conjugation protocols, the new linker s

Full text of DOI:10.1021/bc200069j

ARTICLE
pubs.acs.org/bc
DNA Gold Nanoparticle Conjugates Incorporating
Thiooxonucleosides: 7-Deaza-6-thio-20-deoxyguanosine as
Gold Surface Anchor
Frank Seela,*,†,‡ Ping Ding,†,‡ and Simone Budow†
†Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology, Heisenbergstrasse 11,
48149 M€unster, Germany
‡Laboratorium f€ur Organische und Bioorganische Chemie, Institut f€ur Chemie, Universit€at Osnabr€uck, Barbarastrasse 7,
49069 Osnabr€uck, Germany
S Supporting Information
b
ABSTRACT: A new protocol for the covalent attachment of
oligonucleotides to gold nanoparticles was developed. Base-
modified nucleosides with thiooxo groups were acting as mo-
lecular surface anchor. Compared to already existing conjugation
protocols, the new linker strategy simplifies the synthesis of
DNA gold nanoparticle conjugates. The phosphoramidite of
7-deaza-6-thio-20-deoxyguanosine (6) was used in solid-phase
synthesis. Incorporation of the sulfur-containing nucleosides can
be performed at any position of an oligonucleotide; even multi-
ple incorporations are feasible, which will increase the binding stability of the corresponding oligonucleotides to the gold
nanoparticles. Oligonucleotide strands immobilized at the end of a chain were easily accessible during hybridization leading to
DNA gold nanoparticle network formation. On the contrary, oligonucleotides immobilized via a central position could not form a
DNA-AuNP network. Melting studies of the DNA gold nanoparticle assemblies revealed sharp melting profiles with a very narrow
melting transition.
’ INTRODUCTION
strong affinity to the noble metal gold. There is still a contro-
versial discussion on the mechanism of immobilization. How-
ever, an X-ray structure of a thiol monolayer protected gold
nanoparticle has been reported recently.16 Further developments
in the field of DNA-AuNPs include the incorporation of sulfur
functionalities into the oligonucleotide chain as an alternative to
the conventional alkylthiol linker.17,18 Phosphorothioate-mod-
ified DNA was utilized for gold nanoparticle conjugation either
by directly coupling the DNA backbone to AuNPs19 or by using a
short linker molecule connecting the DNA backbone and the
AuNPs.20
It is worth mentioning that tRNAs contain a large diversity of
modified nucleosides;21 a few of them bear thio functions at the
nucleobase. Examples are 2-thiouridine, 4-thiouridine, or 2-thio-
cytidine including derivatives with various substituents in the
5-position. Nucleosides with thiooxo groups have already been
incorporated into artificial DNA, either chemically or by
polymerases.22ꢀ25 Consequently, we initiated a study to use
these naturally occurring modification sites for oligonucleotide
immobilization on gold nanoparticles.
DNA guided assemblies of metal nanoparticles have great
potential for advanced material applications, in biomedical
sciences, and as diagnostics tools.1,2 DNA nanoparticle conjugates
have been employed in nucleic acid detection and labeling and
were utilized as barcodes, biosensors, or carriers for the delivery of
DNA.1ꢀ6 Additionally, they can be used for the bottom-up
assembly of nanoarchitectures.7,8 Among the accessible metal
nanoparticles, gold nanoparticles (AuNPs) gained particular
attention due to their chemical inertness and the ease of surface
modification.1,9,10 DNA gold nanoparticle (DNA-AuNP) conju-
gates combine the favorable properties of gold colloids with those
of DNA. A number of key advantages for using the DNA molecule
as a “construction material” are (i) its unique molecular recogni-
tion properties, (ii) itseasy access by automated DNAsynthesis or
enzymatic polymerization, and (iii) its self-assembly capacities to
form multistranded aggregates allowing the construction of DNA-
based nanoscaled devices.7,11
To meet the above-mentioned applications, the conjugates
must be stable in aqueous buffer systems, even at elevated
temperature. For this, several strategies were developed for
preparing DNA-AuNP conjugates.12ꢀ15 The common protocol
employs oligonucleotides with 30 or 50-terminal thiol groups
introduced by linker units. The covalent attachment to AuNPs
proceeds via the thiol functionality thereby making use of its
Received: February 3, 2011
Revised:
March 8, 2011
Published: March 28, 2011
r
2011 American Chemical Society
794
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Scheme 1. Reaction Route for the Formation of Oligonucleotide AuNP Conjugates with Thionucleoside 1 as Anchor Molecule
and Proposed Conjugate Structure
Figure 1. Thionucleosides 1ꢀ4 employed in this study.
Figure 2. Phosphoramidite building blocks of the hexylthiol linker and the thionucleosides employed for oligonucleotide synthesis.
Herein, we report on a new strategy for conjugation of
DNA with gold nanoparticles. In a first step, oligonucleotides
are synthesized bearing a thiooxo function (“thio” anchor)
at the nucleobase; in a second step, these oligonucleotides
are directly attached to the gold surface without prior sulfur
protection (Scheme 1). From that background, we selected
the four nucleosides 7-deaza-6-thio-20-deoxyguanosine (1),
6-thio-20-deoxyguanosine (2), 4-thio-20-deoxythymidine (3),
and 2-thio-20-deoxythymidine (4) for our study as shown
in Figure 1. They were introduced into oligonucleotides via
phosphoramidite chemistry to become an integer part of single-
stranded oligonucleotides.
The thiooxo group was utilized as a molecular anchor to gold
nanoparticles. First, the stability of the different thiooxonucleo-
sides toward the conditions of solid-phase oligonucleotide
synthesis was investigated, and conjugation to the gold surface
was tested. Hybridization experiments employing DNA gold nano-
particle conjugates proved the formation of stable DNA-AuNP
hybrids with thionucleosides, similar to those linked by conven-
tional protocols.
’ EXPERIMENTAL PROCEDURES
General Methods and Materials. All chemicals were pur-
chased from Acros, Aldrich, Sigma, or Fluka. The 50-alkylthiol
modifier 6-(triphenylmethyl)-S-(CH2)6-O-2-cyanoethyl diiso-
propylphosphoramidite (5) and phosphoramidites 7ꢀ9 were
obtained from Glen Research (Virginia, USA) (Figure 2). 4-tert-
Butylphenoxyacetyl protected canonical phosphoramidites were
purchased from Millipore (Massachusetts, USA). Solvents were
of laboratory grade. Thin-layer chromatography (TLC) was
performed on TLC aluminum sheets covered with silica gel 60
F254 (0.2 mm, VWR International, Germany). Flash column
chromatography (FC): silica gel 60 (VWR International, Darm-
stadt, Germany) at 0.4 bar; UV-spectra were recorded on a
U-3000 spectrophotometer (Hitachi, Japan); λmax (ε) in nm,
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ε in dm3 molꢀ1 cmꢀ1. NMR spectra: DPX 300 spectrometer
H-40), 4.36ꢀ4.37 (m, 1H, H-30), 4.94 (t, J = 5.4 Hz, 1H, HO-
50), 5.00 (s, 2H, Pac-CH2), 5.32 (d, J = 5.3 Hz, 1H, HO-30),
6.54ꢀ6.59 (m, 2H, H-5, H-10), 6.93ꢀ6.98 (m, 3H, phenoxy),
7.28ꢀ7.33 (m, 2H, phenoxy), 7.65 (d, J = 3.9 Hz, 1H, H-6),
10.69 (s, 1H, CO-NH).
1
(Bruker, Germany) at 300 MHz for H, 75 MHz for 13C and
121.5 MHz for 31P. The J values are given in Hz; δ values in
ppm relative to Me4Si as internal standard, or 85% H3PO4 for
31P. For NMR spectra recorded in DMSO, the chemical shift of
1
the solvent peak was set to 2.50 ppm for H NMR and 39.50
4-[(2-Cyanoethyl)thio]-7-[2-deoxy-5-O-(4,40-dimethoxytri-
tyl)-β-D-erythro-pentofuranosyl]-2-phenoxyacetamino-7H-py-
rrolo[2,3-d]pyrimidine (12). Compound 11 (469.5 mg, 1.00
mmol) was coevaporated with anhydrous pyridine (3 ꢁ 5.0 mL)
and then dissolved in pyridine (5.0 mL). To this solution,
4,40-dimethoxytriphenylmethyl chloride (440.5 mg, 1.30 mmol)
was added and the mixture was stirred at r.t. for 3 h. The reaction
was quenched by the addition of MeOH and the mixture was
evaporated to dryness. The mixture was subjected to FC (column
4 ꢁ 10 cm, elution with CH2Cl2/acetone, 20:1) to give 12 as a
colorless foam (555.8 mg, 72%). TLC (CH2Cl2/MeOH, 95:5):
Rf 0.61. UV λmax (MeOH)/nm 302 (ε/dm3 molꢀ1 cmꢀ1
ppm for 13C NMR. Elemental analyses were performed by the
Mikroanalytisches Laboratorium Beller, G€ottingen, Germany.
MALDI-TOF mass spectra were recorded in the linear negative
mode with an Applied Biosystems Voyager DE PRO spectro-
meter with 3-hydroxypicolinic acid (3-HPA) as a matrix. Re-
versed-phase HPLC was carried out on a 4 ꢁ 250 mm RP-18 (10
mm) LiChrospher 100 column (VWR International) with a
Merck-Hitachi HPLC pump (model L-6250) connected with a
variable wavelength monitor (model 655-A), a controller (model
L-500), and an integrator (model D-2500). Melting curves were
measured with a Cary-100 Bio UVꢀvis spectrophotometer
(Varian, Australia) equipped with a Cary thermoelectrical con-
troller with a heating rate of 1 °C/min. The Tm values were
calculated by Meltwin 3.0 program. Nanopure water (resistance
<0.055 μS/cm) from MembraPure water system (Astacus) was
used for all experiments.
1
14 400), 240 (53 500). Anal. (C43H41N5O7S) C, H, N. H
NMR (300 MHz, DMSO-d6): δ = 2.27ꢀ2.31 (m, 1H, HR-20),
2.55ꢀ2.64 (m, 1H, Hβ-20), 3.13ꢀ3.17 (m, 4H, CH2ꢀCN, H-50,
H-500), 3.52ꢀ3.56 (m, 2H, CH2ꢀS), 3.71 (s, 6H, OCH3), 3.95
(m, 1H, H-40), 4.37 (m, 1H, H-30), 5.00 (s, 2H, Pac-CH2), 5.37
(d, J = 4.2 Hz, 1H, HO-30), 6.52ꢀ6.56 (m, 2H, H-5, H-10),
6.78ꢀ7.35 (m, 18H, phenoxy), 7.46 (d, J = 3.6 Hz, 1H, H-6),
10.69 (s, 1H, CO-NH).
7-(2-Deoxy-β-D-erythro-pentofuranosyl)-2-phenoxyacetami-
no-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-thione (10). Compound
1
26 (1.6 g, 5.50 mmol) was dried by repeated coevaporation with
anhydrous pyridine (3 ꢁ 5 mL), then suspended in pyridine
(5 mL). To this solution, trimethylsilyl chloride (3.6 mL, 28.17
mmol) was added at room temperature. After 1 h, phenoxyacetyl
chloride (1.1 mL, 7.96 mmol) was introduced, and the
solution was stirred at room temperature for another 4 h. The
mixture was cooled to 0 °C, diluted with H2O (5 mL), and stirred
for 10 min. Then, aq. ammonia (5 mL) was added, and the
solution was stirred for an additional 15 min. The solvent was
evaporated to dryness. Purification by FC (silica gel, column 8 ꢁ
15 cm, CH2Cl2/MeOH, 50:1f25:1) gave 10 as a colorless foam
(1.9 g, 81%); mp 205 °C. TLC (CH2Cl2/MeOH, 9:1): Rf 0.53.
UV λmax (MeOH)/nm 333 (ε/dm3 molꢀ1 cmꢀ1 47 600). Anal.
(C19H20N4O5S) C, H, N. 1H NMR (300 MHz, DMSO-d6): δ =
2.15ꢀ2.23 (m, 1H, HR-20), 2.38ꢀ2.47 (m, 1H, Hβ-20),
3.51ꢀ3.54 (m, 2H, H-50), 3.81ꢀ3.84 (m, 1H, H-40), 4.33ꢀ4.36
(m, 1H, H-30), 4.88 (s, 2H, Pac-CH2), 4.95 (t, J = 5.3 Hz, 1H,
HO-50), 5.30 (d, J = 3.6 Hz, 1H, HO-30), 6.41 (m, 1H, H-10), 6.64
(d, J = 3.7 Hz, 1H, H-5), 6.96ꢀ7.01 (m, 3H, phenoxy), 7.29ꢀ7.34
(m, 2H, phenoxy), 7.49 (d, J = 3.7 Hz, 1H, H-6), 11.99 (br s, 1H,
NꢀH), 12.89 (br s, 1H, CO-NH).
4-[(2-Cyanoethyl)thio]-7-[2-deoxy-5-O-(4,40-dimethoxytrityl)-
β-D-erythro-pentofuranosyl]-2-phenoxyacetamino-7H-pyrrolo-
[2,3-d]pyrimidine 30-[(2-cyanoethyl)-N,N-diisopropylphospho-
ramidite] (6). Compound 12 (555.8 mg, 0.72 mmol) was
dissolved in anhydrous CH2Cl2 (3.0 mL) under argon and was
reactedwith (2-cyanoethyl)diisopropylphosphoramido chloridite
(225 μL, 0.95 mmol) in the presence of iPr2EtN (220 μL, 1.27
mmol) at room temperature. After 20 min, the reaction mixture
was diluted with CH2Cl2 and the solution was washed with a 5%
aqueous NaHCO3 solution, followed by brine. The organic
solution was dried over anhydrous Na2SO4, filtered, and con-
centrated. The residue was subjected to FC (column 4 ꢁ 10 cm,
CH2Cl2/acetone, 25:1) yielding 6 as a colorless foam (429.1 mg,
61%). TLC (CH2Cl2/acetone, 95:5): Rf 0.64. 31P NMR (121.5
MHz, CDCl3): δ = 148.56; 148.69.
Hydrolytic Stability of Thiooxonucleosides. The hydrolytic
stability of thiooxonucleosides 1ꢀ4 and 11 under standard
deprotection conditions (25% aqueous NH3, 14ꢀ16 h, 60 °C)
was monitored by reversed-phase HPLC (RP-18, 250 ꢁ 4 mm).
Nucleosides 1ꢀ4 and 11 (about 1 mg) were dissolved in 1 mL
of 25% aq. ammonia in a sealed vessel and incubated at 60 °C.
After 16 h incubation, aq. ammonia was removed by evapora-
tion and the residue was redissolved in 1 mL HPLC buffer A.
50 μL aliquots of each sample were injected into the HPLC, and
the spectra were recorded at 260 nm (for spectra see Supporting
Information).
Synthesis, Purification, and Characterization of Oligonu-
cleotides. The syntheses of oligonucleotides were performed on
a DNA synthesizer, model 392ꢀ08 (Applied Biosystems, Wei-
terstadt, Germany) at 1 μmol scale. For oligonucleotides
containing 7-deaza-6-thio-20-deoxyguanosine (1), 2-thio-20-
deoxythymidine (4), or the hexylthiol linker, the corresponding
phosphoramidites 5, 6, and 9 were used, and the standard
synthesis protocol for 30-(2-cyanoethyl phosphoramidites) was
followed.27 The average coupling yield was always higher than
95%. Oligonucleotides containing the hexylthiol linker, 1 or 4,
were cleaved from solid support with 25% aq. NH3 and
4-[(2-Cyanoethyl)thio]-7-(2-deoxy-β-D-erythro-pentofurano-
syl)-2-phenoxyacetamino-7H-pyrrolo[2,3-d]pyrimidine (11).
3-Bromopropionitrile (4.0 mL, 48.07 mmol) and anhydrous
K2CO3 (3.0 g, 21.71 mmol) were added to 25 mL dry DMF and
the mixture was stirred vigorously. Compound 10 (1.9 g, 4.45
mmol) was dissolved in 5 mL dry DMF and added dropwise to
the stirred solution within 30 min. The reaction mixture was
stirred overnight until completion of the reaction (monitored
by TLC). The solvent was removed by coevaporation with
xylene and the residue was applied to FC (silica gel, column 8
ꢁ 15 cm, CH2Cl2/MeOH, 500:1f100:1). The main zone
yielded 11 as a colorless solid (1.2 g, 55%). TLC (CH2Cl2/
MeOH, 95:5): Rf 0.22. UV λmax (MeOH)/nm 301 (ε/dm3
molꢀ1 cmꢀ1 26 700), 244 (73 300). Anal. (C22H23N5O5S) C,
H, N. 1H NMR (300 MHz, DMSO-d6): δ = 2.18ꢀ2.25 (m, 1H,
HR-20), 2.50 (m, 1H, Hβ-20), 3.16 (t, 2H, CH2ꢀCN),
3.51ꢀ3.56 (m, 4H, H-50, H-500, CH2ꢀS), 3.84ꢀ3.85 (m, 1H,
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deprotected for 14ꢀ16 h at 60 °C (standard deprotection
conditions). Oligonucleotides containing 6-thio-20-deoxyguano-
sine (2) and 4-thio-20-deoxythmidine (3) were prepared using the
corresponding phosphoramidites 7 and 8, and reagents required
for the fast deprotection procedure.28 For that, 4-tert-butylphe-
noxyacetyl protected canonical phosphoramidites and the cap-
ping reagent 4-tert-butylphenoxyacetic anhydride instead of acetic
anhydride were used. The coupling efficiency was always higher
than 95%. Oligonucleotides incorporating 2 or 3 were cleaved
from solid support and deprotected in 25% aq. NH3 solution
containing 50 mM NaSH at room temperature overnight
(recommended conditions of the supplier). The DMT-contain-
ing oligonucleotides were purified on reversed-phase HPLC in
the DMT-on modus (Merck-Hitachi-HPLC; RP-18 column)
with the following gradient system [A: 0.1 M (Et3NH)OAc
(pH 7.0)/MeCN 95:5; B: MeCN]: 3 min, 20% B in A, 12 min,
20ꢀ50% B in A and 25 min, 20% B in A, flow rate 1.0 mL/min.
The solvent was evaporated, and the residue was treated with
2.5% CHCl2COOH/CH2Cl2 (400 μL) for 5 min at 0 °C to
remove the 4,40-dimethoxytrityl residue. The detritylated oligo-
mers were purified again by reversed-phase HPLC [gradient:
0ꢀ20 min 0ꢀ20% B in A; flow rate 1 mL/min]. The oligomers
were desalted on a short column (RP-18, silica gel) using H2O for
elution of the salt, while the oligomers were eluted with MeOH/
H2O (3:2). The oligonucleotides were lyophilized on a Speed-
Vac evaporator to yield colorless solids which were frozen
at ꢀ24 °C. The 50-alkylthiol-modified oligonucleotides 50-d(trityl-
S-(CH2)6-T10-TAG GTC AAT ACT) (40) and 50-d(trityl-
S-(CH2)6-T10-AGT ATT GAC CTA) (41) were only purified
in the DMT-on modus by reversed-phase HPLC (RP-18) as
described above.
Table 1. Gold Nanoparticle Conjugates Immobilizing an
Arbitrary Number of Oligonucleotides and the Maximum of
VIS Absorptiona
The enzymatic hydrolysis of the oligonucleotides was per-
formed as described29 with snake venom phosphodiesterase (EC
3.1.15.1, Crotallus adamanteus) and alkaline phosphatase (EC
3.1.3.1, Escherichia coli from Roche Diagnostics GmbH, Germany)
in 0.1 M Tris-HCl buffer (pH 8.3) at 37 °C. The hydrolysis
product was analyzed by reversed-phase HPLC (RP-18). Quanti-
fication of the constituents was made on the basis of the peak areas,
which were divided by the extinction coefficients ε260 of the
nucleosides: dA 15400, dC 7300, dG 11700, dT 8800, 7-deaza-
6-thio-20-deoxyguanosine (1) 10100, 6-thio-20-deoxyguano-
sine (2) 7300, 4-thio-20-deoxythymidine (3) 1500. MALDI-
TOF mass spectra were recorded in the linear negative mode
with an Applied Biosystems Voyager DE PRO spectrometer
with 3-hydroxypicolinic acid (3-HPA) as a matrix. The de-
tected masses were identical with the calculated values (see
Supporting Information).
General Procedure for the Preparation of Oligonucleotide
Gold Nanoparticle Conjugates Employing Thiooxonucleo-
sides as Molecular Anchor. The gold nanoparticle solution
(15 nm particle diameter) was prepared from a HAuCl4 solution
by citrate reduction according to the protocol originally reported
by Turkevitch30 and later described by Letsinger and Mirkin.13
The nanoparticle concentration was determined by UV/vis using
ε520 = 4.2 ꢁ 108 Mꢀ1 cmꢀ1 (UV/vismax: 520 nm).31 The gold
nanoparticles (∼3 nM) were functionalized with oligonucleo-
tides 18ꢀ29 and 36ꢀ39 containing thiooxonucleosides 1ꢀ4 at
different positions within their sequences. The DNA-AuNP
conjugates Au18-Au29 and Au36-Au39 were prepared by mix-
ing 1 mL of the respective AuNP solution with the aq. solution of
the purified oligonucleotide (1ꢀ5 μL) to yield a final oligonu-
cleotide concentration of 3 μM. The coupling reaction was
a Spacer T10 = 50-d(TTT TTT TTT T).
performed at slightly elevated temperature (40 °C). After stand-
ing for 20 h, 5 μL of a 2 M NaCl, 0.2 mM phosphate buffer
solution (pH 7.0) was added under constant stirring to bring the
colloidal solutions to a 0.01 M NaCl concentration. The solution
was allowed to stand for 6ꢀ8 h. The NaCl concentration was
increased stepwise using 2 M NaCl and 0.2 mM phosphate buffer
solution (pH 7.0). First, the colloids were salted to 0.02 M NaCl
and allowed to age for another 6ꢀ8 h, then salted to 0.05 M NaCl
with 6ꢀ8 h of incubation, and were finally salted to 0.1 M NaCl.
Subsequently, the DNA gold nanoparticle solutions were cen-
trifuged (8000 rpm, 45 min) and the clear supernatant was taken
off to remove unbound oligonucleotides. The precipitate was
redispersed in 1 mL of a 0.1 M NaCl, 10 mM phosphate buffer
solution (pH 7.0). After incubation (24 h, 40 °C), the conjugate
solutions were centrifuged and washed again with the same buffer,
finally yielding 1 mL of the DNA-AuNP conjugates Au18-Au29
and Au36-Au39 (Table 1). The DNA-AuNP conjugates were
characterized by UV/vis spectroscopy and stored at 4 °C.
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Scheme 2. Synthesis of Phosphoramidite Building Block 6a
a Reagents and conditions: (i) Me3SiCl, phenoxyacetyl chloride, pyridine, aq. NH3, 4 h, r.t.; (ii) 3-bromopropionitrile, anh. K2CO3, DMF, overnight, r.t.;
(iii) DMTr-Cl, pyridine, 3 h, r.t.; (iv) (2-cyanoethyl)diisopropylphosphoramido chloridite, N,N-diisopropylethylamine, anh. CH2Cl2, 20 min, r.t.
All conjugates show plasmon resonance at around 524 nm indicating
a nonaggregated state (Table 1 and Supporting Information).
Preparation of DNA-AuNP Conjugates Employing the
Hexylthiol Linker As Anchoring Site. DNA-AuNPs employing
oligonucleotides with terminal alkylthiol linkers were prepared
by a protocol reported previously.13,32 The DNA-AuNP con-
jugates Au40 and Au41 were characterized by UV/vis spectros-
copy showing plasmon resonance at 523 nm which is correlated
to a nonaggregated state (for spectra, see Figure S2, Supporting
Information).
functionality was used to attach the DNA oligonucleotides to gold
electrodes.35,36 For our study, we selected the thiooxonucleosides
1ꢀ4 for incorporation into oligonucleotides via phosphoramidite
chemistry. In this respect, the stability of 1ꢀ4 against nucleophilic
sulfur displacement under alkaline deprotection conditions was
investigated. Thiooxonucleosides 1ꢀ4 were used as a molecular
anchor to the gold nanoparticle surface and their capability to
form DNA-AuNPs was evaluated.
Building Block Synthesis. The phosphoramidites 5ꢀ9 were
selected for oligonucleotide syntheses (Figure 2). Compounds 5
and 7ꢀ9 are commercially available, while phosphoramidite 6
has to be synthesized using nucleoside 1 as starting material.26
For the protection of the amino group, the phenoxyacetyl
residue37 was chosen, and the protocol of transient protection
(f 10, 81% yield) was used (Scheme 2). The thiooxo function
was protected with the cyanoethyl group (f 11, 55% yield) as
described for the parent purine compound 2.38 The protected
intermediate 11 was converted into the 50-O-DMT derivative 12
under standard conditions. Phosphitylation with 2-cyanoethyl-
N,N diisopropyl-phosphoramidochloridite furnished the phos-
phoramidite 6 (61% yield, Scheme 2).
Scanning Electron Microscopy (SEM). A typical sample was
prepared by dropping a DNA-AuNP conjugate into a silicon
wafer which was then subsequently dried and imaged.
’ RESULTS AND DISCUSSION
The conventional protocol for the attachment of oligonucleo-
tides onto gold nanoparticle surfaces makes use of oligonucleo-
tides modified at their 50- or 30-termini by an alkylthiol
linker.5,12ꢀ14,32ꢀ34 However, this protocol is laborious and
accompanied by drawbacks to form SH-modified oligonucleo-
tides ready for immobilization. This includes (i) the detritylation
of the protected thiol-group with silver nitrate, (ii) removal of
excess silver nitrate and (iii) desalting of the free thiol-modified
oligonucleotides. Furthermore, the immobilization of the reac-
tive oligonucleotides to the gold nanoparticles has to be per-
formed shortly after preparation to reduce unwanted side
reactions (e.g., disulfide formation). Moreover, silver ions or
metallic silver which can bind to both oligonucleotides and gold
nanoparticles are difficult to remove quantitatively.
In this study, we made use of the sulfur atom of thiooxonucleo-
sides to conjugate oligonucleotides to gold nanoparticles. The
sulfur atom of these nucleosides is expected to bind to the gold
surface via the thiooxo group or the corresponding mercapto
group. Recently, the triphosphate of 4-thio-20-deoxythymidine
was incorporated enzymatically into DNA, and the sulfur
All compounds were characterized by UV-spectra and 1H- and
13C NMR spectra as well as by elemental analysis (Table 2 and
Experimental Procedures section). The assignments of 13C NMR
chemical shifts of the sugar moiety and the protecting groups were
13
made on the basis of 1Hꢀ C gated-decoupled spectra (for more
data, see Supporting Information) in combination with already
published data.38 For 7-deaza-6-thio-20-deoxyguanosine (1), the
chemical shifts for C2 and C6 were only assigned tentatively.38
Therefore, now we used the deuterium isotope upfield shift
approach to assign the carbon-2 signal,39,40 which is directly
connected with the amino group. In a DMSO solution, containing
a H2O/D2O mixture, the carbon-2 signal is split into two singlets
(Figure S6, Supporting Information): the original one and one
which is shifted 0.04 ppm upfield. Consequently, C2 was assigned
to the 152.2 ppm signal and C6 to the 175.7 ppm signal (Table 2).
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Table 2. C-NMR Chemical Shifts (δ) of 7-Deaza-6-thio-20-deoxyguanosine Derivativesa
13
C(2)b
C(2)c
C(4)b
C(6)c
C(4a)b
C(5)c
C(5)b
C(7)c
C(6)b
C(8)c
C(7a)b
C(4)c
compd
C(10)
C(20)
39.5
C(30)
C(40)
C(50)
c7Gd
152.5
152.2
145.4
151.0
151.1
158.5
175.7
174.8
167.3
167.3
100.1
113.1
116.8
119.5
119.4
102.1
104.4
104.7
99.5
116.7
120.2
122.7
125.0
124.8
150.5
147.1
143.4
149.2
149.2
82.2
82.2
82.5
82.6
82.7
70.8
70.9
70.6
70.9
70.7
86.9
87.1
87.1
87.4
85.5
61.9
61.9
61.5
61.8
64.2
e
d
1
-
d
10
11
12
-
d
-
d
99.5
-
a Measured in DMSO-d6 at 298 K. b Systematic numbering. c Purine numbering. d Superimposed by the DMSO signal. c7Gd: 7-deaza-20-
e
deoxyguanosine; 13C NMR chemical shifts taken from ref 41.
Thus, nucleosides 1ꢀ4 and 11 were incubated in 25% aq.
NH3, 16 h, 60 °C (standard oligonucleotide deprotection con-
ditions) and analyzed by reversed-phase HPLC at 260 nm.
Byproducts were identified by coelution with authentic nucleo-
sides (for experimental procedure and HPLC profiles, see the
Supporting Information).
The thiooxo group at position-6 of purine nucleosides or
position-2 of pyrimidine nucleosides is considerably more stable
compared to position-4 of pyrimidine nucleosides. Accordingly,
among nucleosides 1ꢀ4, the thiooxo group of 4-thio-20-deox-
ythymidine (3) is the most labile one toward the standard
oligonucleotide deprotection conditions (32% conversion to
5-methyl-20-deoxycytidine, 15). On the contrary, the thiooxo
group of 2-thio-20-deoxythymidine (4) was the most stable one,
and less than 1% conversion into an unidentified product was
detected (Figure 3 and Supporting Information). The thiooxo
groups of the purine and 7-deazapurine nucleosides 1 and 2
showed similar stabilities (1: 7% conversion, 2: 9% conversion,
Figure 3). For the protected compound 11, conversion into an
unidentified side product amounts to 11% which is within the
same range as for nucleosides 1 and 2. However, it should be
noted that, for 11, the stability of the sulfur function is related to
deprotection and conversion from 6-cyanoethyl(thio) into the
desired 6-sulfur group (Figure 3 and Supporting Information).
From the above-described experiments, the following order of
stability of the thiooxo groups of nucleosides 1ꢀ4 and 11 toward
alkaline hydrolysis (25% aq. NH3, 16 h, 60 °C) was deduced: 4 >
1 > 2 > 11 > 3 (Figure 3).
Figure 3. Graph showing the alkaline hydrolysis stability of thiooxo-
nucleosides 1ꢀ4 and protected 11. After incubation in a solution of 25%
aq. NH3 at 60 °C for 16 h, compounds 1ꢀ4 and 11 were analyzed by
reversed-phase HPLC at 260 nm. Relative quantification was calculated
on the basis of peak areas which were divided by the extinction
coefficient (ε260) of the respective nucleoside (for details see Supporting
Information). s6c7Gd: 7-deaza-6-thio-20-deoxyguanosine (1), a2c7Ad:
2-amino-7-deaza-20-deoxyadenosine (13), s6Gd: 6-thio-20-deoxyguano-
sine (2), a2Ad: 2-amino-20-deoxyadenosine (14), s4Td: 4-thio-20-deox-
ythymidine (3), m5Cd: 5-methyl-20-deoxycytidine (15), s2Td: 2-thio-20-
deoxythymidine (4), 11: 6-[(2-cyanoethyl)thio]-2-phenoxyacetamino-
7-deaza-20-deoxyguanosine.
Oligonucleotide Synthesis and Characterization. With the
informations of the alkaline hydrolysis experiments in hand, a
series of unmodified and modified oligonucleotides (16ꢀ39)
containing thiooxonucleosides 1ꢀ4 at different positions were
synthesized on solid phase at 1 μmol scale (Table 4), and the
accessibility of pure oligomers was studied. Two different pro-
tocols of oligonucleotide syntheses were employed: (i) oligonu-
cleotide synthesis using fast deprotectable building blocks and
(ii) standard oligonucleotide synthesis employing standard
building blocks. Oligonucleotides incorporating 4-thio-20-deox-
ythymidine (3) or 6-thio-20-deoxyguanosine (2) were synthe-
sized under conditions already reported in the literature utilizing
phosphoramidites 7 and 8 which are suitable for fast deprotec-
tion conditions.38,43 Oligonucleotides were cleaved from solid
support and deprotected in a 25% aq. NH3 solution containing
50 mM NaSH at room temperature overnight. Sulfur exchange
was avoided by employing the protocol of fast deprotection28
and the presence of sodium hydrogensulfide. In contrast, oligo-
nucleotides containing 2-thio-20-deoxythmidine (4) or 7-deaza-
6-thio-20-deoxyguanosine (1) were synthesized and deprotected
Moreover, compared to the parent nucleoside 7-deaza-20-deox-
yguanosine (c7Gd; Table 2), the sulfur atom of thionucleoside 1
attached to carbon-6—instead of the oxygen atom of c7Gd—
induces a significant downfield shift of 17.2 ppm on C6.
Stability of Thiooxonucleosides 1ꢀ4 toward Alkaline
Hydrolysis. Prior to oligonucleotide synthesis, the stability of
the thiooxo group of nucleosides 1ꢀ4 toward the standard
oligonucleotide deprotection conditions (25% aq. NH3, 16 h,
60 °C) was investigated. This study was motivated by earlier
reports on sulfur-amino group exchanges of 6-thio-20-deoxygua-
nosine (2) (f 2-amino-20-deoxyadenosine, 14)24 and 4-thio-20-
deoxythymidine (3) (f 5-methyl-20-deoxycytidine, 15)42 under
alkaline conditions. Moreover, the unknown compound 11
employing a cyanoethyl protecting group at the sulfur function
and a 2-phenoxyacetamino group was also subject of this study.
With this experiment, we intended to confirm the applicability of
the cyanoethyl protecting group at sulfur-6 under standard
oligonucleotide deprotection conditions and conversion to the
desired 7-deaza-6-thio-20-deoxyguanosine (1).
799
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ARTICLE
Figure 4. HPLC elution profiles of (a) the enzymatic hydrolysis products of oligonucleotide 30 obtained after hydrolysis with snake venom
phosphodiesterase and alkaline phosphatase in 0.1 M TrisꢀHCl buffer (pH 8.5) at 37 °C and (b) the artifical mixture of the hydrolysis products of
oligonucleotide 30 and thiooxonucleoside 1. Column: RP-18 (200 ꢁ 10 mm); gradient [A: 0.1 M (Et3NH)OAc (pH 7.0)/MeCN 95:5]: 100% A; flow
rate: 0.7 mL/min.
under standard conditions (25% aq. NH3, 14ꢀ16 h, 60 °C) in the
absence of sodium hydrogensulfide. The oligonucleotides were
isolated and purified by reversed-phase HPLC (for details see
Experimental Procedures).
The nucleobase composition of the modified oligonucleotides
was also confirmed by tandem enzymatic hydrolysis with snake-
venom phosphodiesterase (EC 3.1.15.1, Crotallus adamanteus)
followed by alkaline phosphatase (EC 3.1.3.1, Escherichia coli) in
0.1 M Tris-HCl buffer (pH 8.5) at 37 °C as described
previously.29 As an example, the HPLC profiles of the nucleoside
composition of oligonucleotide 30 after enzymatic hydrolysis
(Figure 4a) and the artificial mixture of the hydrolysis products of
30 and nucleoside 1 (Figure 4b) are shown depicting the correct
nucleobase composition of the oligonucleotide.
Both MALDI-TOF mass spectrometry (Supporting Informa-
tion, Table S1) and enzymatic hydrolysis confirm that deprotec-
tion of oligonucleotides containing 1 under standard conditions
Figure 5. UV/vis profiles of selected DNA-AuNP conjugates in 0.1 M
(25% aq. NH3, 14ꢀ16 h, 60 °C) yields the desired 7-deaza-6-thio-
NaCl, 10 mM phosphate buffer, pH 7.0.
20-deoxyguanosine (1) as constituent of the respective oligonu-
cleotides, as no significant side products pointing toward sulfur
presence of NaCl, UV/vis absorption maximum, and the ability
to hybridize with complementary conjugates, were compared to
DNA-AuNP conjugates utilizing the 50-hexylthiol linker as
molecular anchor.
exchange were detected. These findings make 1 highly advanta-
geous over its purine congener 2 from which severe side product
formation was reported when treated under these conditions.38
A problem associated with oligonucleotides containing thio-
nucleosides is disulfide formation leading to cross-linked oligo-
nucleotides. Manuscripts reporting on interstrand cross-linking
through thionucleosides describe template directed protocols and
the application of mild oxidants to achieve disulfide forma-
tion.44,45 In addition, according to Waters and Connolly,24
disulfide formation is only observed when oligonucleotides are
kept in aqueous solution at neutral pH values at room tempera-
ture. Therefore, to avoid disulfide formation, we always stored our
oligonucleotides as dry material at ꢀ20 °C. Throughout synthe-
sis, purification, and characterization of oligonucleotides incor-
porating thionucleosides 1ꢀ4, we did not make any observations
indicating cross-linking of oligonucleotides due to disulfide for-
mation. As mentioned above, in all cases, the detected masses of
oligonucleotides were identical with the calculated values
(Supporting Information, Table S1). Moreover, it should be
noted that covalent binding to AuNPs can be achieved via thiol
groups as well as disulfides.17,46
The unmodified AuNPs were functionalized with oligonucleo-
tides 18ꢀ39 containing one or multiple thiooxonucleosides
(1ꢀ4) at different positions. For that, 1 mL of the unmodified
AuNP solution was mixed with the aqueous solution of the
respective purified oligonucleotide (1ꢀ5 μL) to yield a final
oligonucleotide concentration of 3 μM. The conjugation was
performed at slightly elevated temperature (40 °C). The NaCl
concentration of the DNA-AuNP solution was increased stepwise
with phosphate buffer (2 M NaCl, 0.2 mM phosphate buffer, pH
7.0) to a final NaCl concentration of 0.1 M. Unbound oligonu-
cleotides were removed from the DNA-AuNP solutions by a
repeated washing and centrifugation protocol with 0.1 M NaCl,
10 mM phosphate buffer (pH 7.0) (for details see Experimental
Procedures). During the whole procedure, the DNA gold nano-
particle solutions stayeddeep redincolor. However, wefound that
the preparation of DNA-AuNP conjugates with oligonucleotides
20ꢀ21 or 24ꢀ25 incorporating 2 and 4 were encountered with
difficulties. Contrary to all other samples, conjugation of 20ꢀ21
and 24ꢀ25 to AuNPs failed several times (50% failure). More-
over, the centrifugation speed had to be reduced significantly
from 8000 rpm (standard speed) to 5800 rpm; otherwise, it was
not possible to redisperse the precipitate. Finally, all DNA-AuNP
Preparation of Oligonucleotide Gold Nanoparticle Con-
jugates. Next, the applicability of thionucleosides 1ꢀ4 as
components of single-stranded oligonucleotides was evaluated
for nanoparticle conjugation. Selected key properties of the
corresponding DNA-AuNP conjugates, such as stability in the
800
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Table 3. Applicability of Thiooxonucleosides 1ꢀ4 and 50-Hexylthiol Linker for Gold Nanoparticle Conjugate Formatione
a Stable oligonucleotide gold nanoparticle conjugates were obtained in 0.1 M NaCl, 10 mM phosphate buffer, pH 7.0. b Stable oligonucleotide gold
nanoparticle conjugates were obtained in 0.2 M NaCl, 10 mM phosphate buffer, pH 7.0. c The hybridization experiment was performed in 0.1 M NaCl,
10 mM phosphate buffer, pH 7.0 with overnight incubation time. d Formation of the DNA gold nanoparticle network occurred much more slowly and
required several days. Spacer T10 = 50-d(TTT TTT TTT T). e Red ball = 15-nm-diameter gold nanoparticle.
conjugates Au18-Au29 and Au36-Au39 showed the expected
plasmon resonance at around 524 nm indicating a nonaggregated
state (Table 1, Figure 5, and Figure S2, Supporting Information).
It is worthwhile to mention that the same absorption maximum
(524 nm) is also observed for DNA-AuNP conjugates Au36-Au39,
in which the attachment succeeds through thionucleoside 1
situated within an internal position of the respective oligonucleo-
tide (Figure 5). Thus, it can be concluded that thionucleoside 1
can also function as a molecular anchor for AuNPs through an
internal position of an oligonucleotide.
Table 4. Tm Values and Thermodynamic Data of Oligo-
nucleotide Duplexes Containing Thiooxonucleosides 1ꢀ4
as Overhanging Ends or Opposite Canonical Nucleosidesa,b
Tm
[°C]
ΔTm
duplex
[°C]c
50-d(TAG GTC AAT ACT)
30-d(ATC CAG TTA TGA)
(16)
(17)
47
48
47
47
47
47
47
43
40
17
65
-
þ1
0
50-d(1 T10 TAG GTC AAT ACT)
30-d(ATC CAG TTA TGA T10 1)
(18)
(19)
For comparison, oligonucleotides 40 and 41 incorporating a 50-
hexylthiol linker were conjugated to AuNPs (f Au40, Au41;
Table 1) employing the conventional protocol reported earlier by
others.47 The UV/vis spectra of DNA-AuNP conjugates Au40 and
Au41 show a plasmon resonance at 523 nm (Table 1, Figure 5).
Next, the stability of DNA-AuNP conjugates Au18-Au29
containing the different thiooxonucleosides 1ꢀ4 and Au40,
Au41 employing the hexylthiol linker was evaluated at higher
salt concentration (0.2 M NaCl, 10 mM phosphate, pH 7.0 buffer
solution). For that, UV/vis spectra of the DNA-AuNP conjugate
solutions were recorded at different time intervals and compared
(for UV/vis profiles at the different time intervals see the
Supporting Information, Figure S3). This experiment was per-
formed to provide more information on the applicability of 1ꢀ4
for AuNP conjugation and hence on the stability of the different
conjugates (Table 3). The DNA-AuNP conjugates Au22-Au25
using 4-thio-20-deoxythymidine (3) or 2-thio-20-deoxythymidine
(4) as anchor molecules were unstable in the presence of higher
NaCl concentration. This became immediately evident for DNA-
AuNPs Au24 and Au25 containing 2-thio-20-deoxythymidine
(4), due to aggregation of the DNA-AuNPs (solutions turned
black) followed by precipitation (Table 3, entry 4), whereas
DNA-AuNPs Au22 and Au23 showed partial precipitation which
was deduced from a significant decrease in the maximum of the
UV/vis absorption profile (36%) after overnight incubation
compared to the original profile (0 h incubation) (Figure S3,
Supporting Information). However, the plasmon resonance of
conjugates Au22 and Au23 was almost unchanged (524.5 nm),
but the red color of the solutions was much fainter compared to
the original solutions. According to these observations, we
classified DNA-AuNP conjugates using 4-thio-20-deoxythymi-
dine (3) as anchor molecule (Au22 and Au23) as not stable in
the presence of higher NaCl concentration (see Table 3, entry 3).
50-d(2 T10 TAG GTC AAT ACT)
30-d(ATC CAG TTA TGA T10 2)
(20)
(21)
50-d(3 T10 TAG GTC AAT ACT)
30-d(ATC CAG TTA TGA T10 3)
(22)
(23)
0
50-d(4 T10 TAG GTC AAT ACT)
30-d(ATC CAG TTA TGA T10 4)
(24)
(25)
0
50-d(11 T10 TAG GTC AAT ACT)
30-d(ATC CAG TTA TGA T10 11)
(26)
(27)
0
50-d(111 T10 TAG GTC AAT ACT)
30-d(ATC CAG TTA TGA T10 111)
(28)
(29)
0
50-d(TAG GTC AAT ACT)
30-d(ATC CAG TTA T1A)
(16)
(30)
ꢀ4
ꢀ7
ꢀ30
-
50-d(TAG GTC AAT ACT)
30-d(ATC CA1 TTA TGA)
(16)
(31)
50-d(TA11TC AAT ACT)
30-d(ATC CA1 TTA T1A)
(32)
(33)
50-d(TAG GTC AAT ACT TAG GTC AAT ACT) (34)
30-d(ATC CAG TTA TGA ATC CAG TTA TGA) (35)
50-d(TA1 GTC AAT ACT TAG GTC AAT ACT)
30-d(ATC CAG TTA TGA ATC CAG TTA T1A)
(36)
(37)
55
53
ꢀ10
ꢀ12
50-d(TAG GTC AAT ACT TA1 GTC AAT ACT)
30-d(ATC CAG TTA T1A ATC CAG TTA TGA)
(38)
(39)
a Measured at 260 nm in 0.1 M NaCl, 10 mM phosphate buffer
(pH 7.0), with
5
μM single-strand concentration. b Spacer
base mismatch
T
ꢀ T
10 = 50-d(TTT TTT TTT T). c ΔTm was calculated as Tm
mbase match
.
801
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Figure 6. UV/vis spectra of (a) DNA-AuNP conjugates Au18, Au19 using 7-deaza-6-thio-20-deoxyguanosine (1) as anchor, and the DNA-AuNP
network Au18•Au19 formed after overnight incubation (blue curve). (b) DNA-AuNP conjugates Au20 and Au21 using 6-thio-20-deoxyguanosine (2)
as anchor (red and black curves), the DNA-AuNP network Au20•Au21 formed after overnight incubation (blue curve) and after several days of
incubation (green curve). (c) DNA-AuNP conjugates Au22 and Au23 using 4-thio-20-deoxythymidine (3) as anchor and the DNA-AuNP network
formed by Au22•Au23 after overnight incubation (blue curve). (d) DNA-AuNP conjugates Au24 and Au25 using 2-thio-20-deoxythymidine (4) as
anchor (red and black curves) and after overnight incubation (blue curve).
On the contrary, the DNA-AuNPs Au18-Au21 using 7-deaza-6-
thio-20-deoxyguanosine (1) or 6-thio-20-deoxyguanosine (2) as
conjugation site (Table 3, entry 1ꢀ2) and Au40, Au41 making
use of a hexylthiol linker (Table 3, entry 5) were of comparable
stability, and all conjugates were significantly more stable than
Au22-Au25 under the higher salt concentration (see also Sup-
porting Information, Figure S3).
Similar results were found for DNA-AuNP conjugates Au26-
Au29 having consecutive 7-deaza-6-thio-20-deoxyguanosine (1)
modifications at the 50-end of oligonucleotides 26ꢀ29, after
overnight incubation in 0.2 M NaCl, 10 mM phosphate, pH 7.0
buffer solution (data not shown).
For clarification, hydridization studies were performed with non-
immobilized oligonucleotide duplexes incorporating compounds
1ꢀ4 (Table 4). Single or multiple thiooxonucleosides (1ꢀ4)
were incorporated as overhanging ends together with a 50-d(T)10
spacer. Table 4 shows that all duplexes have similar Tm values
(47 °C) as the parent unmodified duplex 50-d(TAG GTC AAT
ACT) (16) • 30-d(ATC CAG TTA TGA) (17).
As 7-deaza-6-thio-20-deoxyguanosine (1) has not been incor-
porated into oligonucleotides before, its properties as a consti-
tuent of duplex DNA were investigated in more detail. Thus,
consecutive and random positions have been selected for mod-
ification with 1, and Tm values were determined.
A single replacement of nucleoside 1 in the center or at the
periphery of the 12-mer duplex reduced the stability of the
duplex. The effect was more pronounced when 1 was incorpo-
rated in the center of the oligonucleotide duplex (16•31:
ΔTm = ꢀ7 °C) than at the periphery (16•30: ΔTm = ꢀ4 °C).
Multiple modifications led to a significant further destabilization
(ΔTm = ꢀ30 °C for 32•33) (Table 4). Consistently, two
Duplex Stability of Oligonucleotides Containing Thioox-
onucleosides 1ꢀ4 and Oligonucleotide Gold Nanoparticle
Conjugates. It is well-known that in thiooxonucleosides the
replacement of the oxygen atom by sulfur leads to a destabiliza-
tion of duplex DNA since the thio group is a poorer hydrogen
bond acceptor than the normal carbonyl, and bulkiness of the
sulfur atom can interfere with hybridization.48
802
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ARTICLE
Figure 7. Left cuvette: unmodified deep red gold nanoparticle solution.
Right cuvette: purple DNA-AuNP conjugate solution after assembly of
Au18•Au19 (12 h incubation, the sample is shown after intensive
shaking).
incorporations of nucleoside 1 within the center or at the periphery
of 24-mer duplexes (36•37, 38•39) lead to a strong decrease of the
duplex stability when compared to the parent unmodified duplex
34•35 (ΔTm = ꢀ10 °C for 36•37 and ΔTm = ꢀ12 °C for 38•39).
As can be seen from Table 4, 7-deaza-6-thio-20-deoxyguanosine (1)
has a strong destabilizing effect on duplex stability similar to that
reported for the parent 6-thio-20-deoxyguanosine (2).48
Next, hybridization experiments were performed with oligo-
nucleotide gold nanoparticle conjugates. In a typical experiment,
two DNA-AuNP conjugates carrying oligonucleotides with com-
plementary sequences were mixed together (equal con-
centrations), e.g., DNA-AuNP conjugates Au18 and Au19
using 7-deaza-6-thio-20-deoxyguanosine (1) as molecular anchor.
After incubation overnight at 5 °C, the propensity of hybridiza-
tion was evaluated by UV/vis measurements. Almost all com-
plementary DNA-AuNP conjugates show hybridization
evidenced by slow red shifting and broadening of the plasmon
resonance band (Table 3 and Figures 6, 7) with the exception of
the complementary conjugates Au24 and Au25 which utilize
2-thio-20-deoxythymidine (4) as molecular anchor.
In the case of the complementary conjugates Au18•Au19,
Au22•Au23, and Au40•Au41, hybridization led to the formation of
a DNA gold nanoparticle network which precipitated from solution
(f dark red precipitate with a clear supernatant) within one night.
After intensive shaking of the DNA-AuNP solution, the precipitate
can be redispersed leading to a purple solution with UV/vis maxima
of 567 nm for Au18•Au19 (Figure 6a), 548 nm for Au22•Au23
(Figure 6c), and 554 nm for Au40•Au41 (spectra not shown).
On the contrary, the UV/vis spectrum of the DNA-AuNP
solution containing the complementary conjugates Au20 and
Au21, which use 6-thio-20-deoxyguanosine (2) as molecular
anchor, shows only a moderate red shift (524 nmf538 nm) of
the plasmon resonance band accompanied by a smaller decrease
of the absorption and only a slight broadening of the plasmon
resonance band (Figure 6b, blue curve). However, no pre-
cipitation of the DNA-AuNPs was observed after incubation
overnight. Prolonged incubation of about 1 week finally led to the
precipitation of the DNA gold nanoparticle network. After
intensive shaking of the DNA-AuNP solution Au20•Au21, the
Figure 8. (a) SEM image of DNA-AuNP conjugate Au18 employing
7-deaza-6-thio-20-deoxyguanosine (1) as molecular anchor in 0.1 M
NaCl, 10 mM phosphate buffer (pH 7.0); (b) SEM image of the DNA-
AuNP network formed by complementary conjugates Au18 and Au19
in 0.1 M NaCl, 10 mM phosphate buffer (pH 7.0).
precipitate was redispersed leading to a purple solution with a
UV/vis maximum of 550 nm (Figure 6b, green curve). Although
the experiments was repeated several times, no network forma-
tion was observed for the complementary conjugates Au24 and
Au25 containing 2-thio-20-deoxythymidine (4), even after pro-
longed incubation of 1ꢀ2 weeks. The absorption maximum of
the DNA-AuNP solution of Au24 and Au25 remained un-
changed at around 524 nm (Figure 6d).
Taking together with the results summarized in Table 3, it can
be concluded that, from all tested thionucleosides (1ꢀ4), the
capability of 7-deaza-6-thio-20-deoxyguanosine (1) as compo-
nent of DNA oligonucleotides to form stable DNA gold nano-
particle conjugates is superior to that of thionucleosides 2ꢀ4.
DNA-AuNPs containing 1 are of comparable stability to those
obtained from oligonucleotides containing the hexylthiol linker.
On the contrary, thionucleosides 2ꢀ4 showed different draw-
backs. For thionucleoside 2, the time required for hybridization
was much longer than for 1. Up to now, we do not have an
explanation regarding the prolonged incubation time required
for the DNA-AuNP network formation of DNA-AuNPs with
6-thio-20-deoxyguanosine (2) as molecular anchor. DNA-AuNPs
with 3 and 4 were unstable in the presence of higher NaCl
803
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Table 5. Tm Values of DNA-AuNP Assemblies Incorporating
Thionucleoside 1 and the Hexylthiol Linker.a,b,c
a Measured at 520 nm in 0.1 M NaCl, 10 mM phosphate buffer (pH 7.0),
with A520 = 2.1 for each DNA-AuNP conjugate solution. b Red ball = 15-
nm-diameter gold nanoparticle. c Spacer T10 = 50-d(TTT TTT TTT T).
n.m. no melting observed.
containing the DNA-AuNP networks were heated (15 °C f
75 °C), and the vis absorption change at 520 nm was observed
while stirring the DNA-AuNP solution. The Tm values were
determined by taking the maximum of the first derivative of a
melting transition and are enlisted in Table 5.
A typical sharp melting profile of Au18•Au19 is shown in
Figure 9a indicating a Tm value of 54 °C for the network of DNA-
AuNPs assembled by duplex 18•19. For comparison, the dis-
sociation of the network formed by the conjugates Au40 and
Au41 containing complementary oligonucleotides with hex-
ylthiol linker was investigated under exactly the same conditions
as described for the Au18•Au19 assembly. For Au40•Au41, an
almost identical Tm value of 53 °C was determined (Figure 9a
and Table 5). It should be noted that both assemblies employ the
same sequence consisting of a 12-mer oligonucleotide for base
pairing and a 10-mer poly dT spacer, while the only difference is
the molecular anchor being 7-deaza-6-thio-20-deoxyguanosine
(1) for Au18•Au19 and the hexylthiol linker for Au40•Au41.
Moreover, both assemblies exhibit melting profiles with a very
narrow melting transition (about 4 °C) as demonstrated in
Figure 9a, whereas for the parent free oligonucleotide duplex
16•17, melting occurs over a much broader temperature range
(about 20 °C) (Figure 9a). This finding is consistent with
observations made earlier by others reporting that oligonucleo-
tide duplexes covalently bound to gold nanoparticles show highly
cooperative melting properties of the network forming duplexes,
which is reflected by a sharp melting transition.12,18,49
Figure 9. (a) Melting profile of the free duplex 16•17 determined at
260 nm (blue curve), and melting profiles of the DNA-AuNP assemblies
of Au18•Au19 (red curve) and Au40•Au41 (blue curve) determined at
520 nm. (b) Melting profiles of the DNA-AuNP assemblies of
Au18•Au19 (black curve), Au26•Au27 (red curve), and Au28•Au29
(blue curve) determined at 520 nm.
concentration, and in addition, DNA-AuNPs with 4 are incapable
of hybridization.
Thus, exemplarily, we selected DNA-AuNP solutions contain-
ing 1 to perform scanning electron microscopy (SEM) to
visualize network formation. A representative section of an image
showing the DNA-AuNP network formed by complementary
conjugates Au18 and Au19 containing thionucleoside 1 is
presented in Figure 8b. The SEM image shows the formation
of large assembled networks of DNA-AuNP conjugates. Besides
the assembled network, the neighboring area is remarkably
“empty”. For comparison, SEM images of nonassembled DNA-
AuNP conjugates, such as Au18 (Figure 8a), show small clusters
of DNA-AuNPs which are fairly regularly distributed. The
formation of large assembled networks as in Figure 8b was not
observed in the case of Au18.
On the basis of the above-described results, melting studies
were focused on DNA-AuNP conjugates employing thionucleoside
1 at different positions within the oligonucleotide chain. For
comparison, Tm values were also determined for DNA-AuNP
conjugates utilizing the hexylthiol linker. For this, the solutions
Next, complementary DNA-AuNPs functionalized with multi-
ple 50-terminal thionucleosides 1 were allowed to hybridize (f
Au26•Au27, Au28•Au29). In both cases, the recognition se-
quence of 18•19 was used together with the 50-d(T)10 linker as
described above. As shown in Table 5, these conjugate
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assemblies exhibit very similar Tm values (Au26•Au27: Tm =
50 °C; Au28•Au29: Tm = 51 °C). The melting profiles of
Au26•Au27 and Au28•Au29 also display a narrow melting
transition range (around 4 °C) and are depicted in Figure 9b.
However, compared to the Tm value found for the assembly
Au18•Au19 employing only one thionucleoside 1 per oligonu-
cleotide strand, these values are 3ꢀ4 °C lower but still within the
same range.
The melting behavior of complementary DNA-AuNP con-
jugates carrying 24-mer oligonucleotides which use thionucleo-
side 1 as anchor molecule at internal positions within their
sequence was also investigated (Au36-Au39, Table 5). The
oligonucleotides of Au36-Au39 are composed of a twofold
repeated recognition sequence allowing formation of 24 base
pairs or partial hybridization (12 base pairs).
solid-phase synthesis with phosphoramidites, e.g., with 6, as well as
enzymatic polymerization with corresponding triphosphates em-
ploying polymerases or terminal transferases can be performed.
7-Deaza-6-thio-20-deoxyguanosine (1) can replace dG. The
lower stability of nucleoside 1•dC base pairs compared to those
withdGor 7-deaza-20-deoxyguanosineresultingfromthebulkiness
of the thiooxo group will not affect hybridization when the
incorporation of 1 occurs outside of the recognition sequence.
UV/vis spectra and hybridization properties of DNA-AuNP con-
jugates using thionucleoside 1 as molecular anchor reveal proper-
ties which are comparable to those containing the commercial
hexanethiol linker.
’ ASSOCIATED CONTENT
The DNA-AuNP conjugates Au38 and Au39 employing 24-
mer complementary oligonucleotides with compound 1 located
in the center of each oligonucleotide cannot form a DNA-AuNP
interlinked network. Even prolonged incubation did not lead to a
color change of the combined conjugate solution, and no shift of
the vis maximum was observed. Thus, it can be concluded that no
hybridization occurred, which is due to the negative effect of an
internal thiooxo group on hybridization. Moreover, hybridiza-
tion is probably additionally hindered by the short distance to the
immobilization site of the gold nanoparticles.
S
Supporting Information. MALDI-TOF mass data of
b
oligonucleotides, 1Hꢀ C coupling constants, protocol of alka-
13
line hydrolytic stability of thiooxonucleosides, HPLC profiles of
the hydrolytic stability of thiooxonucleoside, UV/vis spectra of
DNA-AuNP conjugates, UV/vis spectra of DNA-AuNP conju-
gates in high salt buffer, 1H-, 13C-, and 31P NMR spectra of the
synthesized compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
On the contrary, a Tm value of 53 °C was detected for the
closely related assembly Au36•Au37 with thionucleoside 1 being
located at the periphery of the 24-mer oligonucleotide sequences.
This Tm value is consistent with that of Au18•Au19 employing a
complementary 12-mer duplex for assembly. In this regard, the
observed Tm value points to a partial hybridization of the
complementary oligonucleotides within the DNA-AuNP assem-
bly. Two principle situations appear to be most conclusive, (i) a
matched duplex with fraying ends and base pairing only in the
center of the duplex and (ii) a shift of the complementary
sequences with respect to each other leading to partial hybridiza-
tion of the duplex and leaving the unpaired nucleosides as spacers
between the recognition site and the gold nanoparticles. We
think that a partial overlap of the recognition sites are responsible
for the almost identical Tm values of Au36•Au37 and Au18•
Au19. Thus, the second recognition element is acting simply as
spacer extending the distance to the gold nanoparticles not taking
part in the hybridization process.
Corresponding Author
*Corresponding author. Prof. Dr. Frank Seela. Phone: þ49(0)251
53406 500; Fax: þ49(0)251 53406 857. E-mail: Frank.Seela@
uni-osnabrueck.de. Homepage: www.seela.net.
’ ACKNOWLEDGMENT
We would like to thank Dr. Peter Leonard and Dr. Dieter
Heindl for helpful discussions and support, and Mr. Suresh S.
Pujari for critical reading of the manuscript. We also thank Dr. T.
Koch from Roche Diagnostics GmbH, Penzberg, Germany, for
the measurement of the MALDI spectra, Mr. Nhat Quang Tran
for the oligonucleotide syntheses, and Dr. Chongwen Wang for
SEM measurements. Financial support by the Roche Diagnostics
GmbH and ChemBiotech, Muenster, Germany is highly
appreciated.
’ CONCLUSIONS
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