Facile synthesis of hydroxymethylcytosine-containing oligonucleotides and their reactivity upon osmium oxidation

Article abstract of DOI:10.1039/c1ob05247k

DNA strands containing a 5-hydroxymethylcytosine (^hmC), which have recently been found in neuron cells and embryonic stem cells, were synthesized through a facile synthetic technique. The ^hmC-containing strands were efficiently oxidized at ^hmC using an osmium oxidation assay. The ^hmC was oxidized as easily as 5-methylcytosine, which can be distinguished from unmethylated cytosine.

Full text of DOI:10.1039/c1ob05247k

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PAPER
Facile synthesis of hydroxymethylcytosine-containing oligonucleotides and
their reactivity upon osmium oxidation†
Kaori Sugizaki,a Shuji Ikeda,a Hiroyuki Yanagisawaa and Akimitsu Okamoto*a,b
Received 16th February 2011, Accepted 28th March 2011
DOI: 10.1039/c1ob05247k
DNA strands containing a 5-hydroxymethylcytosine (hmC), which have recently been found in neuron
cells and embryonic stem cells, were synthesized through a facile synthetic technique. The
hmC-containing strands were efficiently oxidized at hmC using an osmium oxidation assay. The hmC was
oxidized as easily as 5-methylcytosine, which can be distinguished from unmethylated cytosine.
upon osmium oxidation. The hmC in ODNs showed high reactivity
upon osmium oxidation. The osmium oxidation method gave a
stable ternary complex at hmC in ODNs.
Introduction
5-Hydroxymethylcytosine (hmC) is a newly discovered natural
nucleobase, which is abundant in Purkinje neuron cells1 and
mouse embryonic stem cells.2 hmC is induced by modification of 5-
methylcytosine (mC) with TET proteins, which have potential roles
in epigenetic regulation. Given the critical role of mC in epigenetic
regulation, hmC may have an important biological role in vivo,
such as acting as an intermediate in a pathway for active DNA
demethylation.
To analyze and understand hmC in DNA, effective detection
methods for hmC are required. The bisulfite technique, which
is a conventional method for distinguishing cytosine (C) from
mC,3 has already been reported to show that neither hmC nor
mC in DNA undergo deamination, although it is likely that hmC
was converted into cytosine 5-methylenesulfonate by bisulfite
treatment.4,5 Osmium oxidation is an effective method for mC
detection, whichmakes itpossible to analyze mC inDNAsequence-
selectively and mC-positively in a short time without any DNA
scission.6,7 mC is oxidized efficiently by exposure to a reaction
mixture of potassium osmate, potassium hexacyanoferrate(III),
and a bipyridine ligand, a stable methylcytosine glycol–osmate–
bipyridine complex being formed, making possible a clear distinc-
tion from inefficient oxidation of unmethylated cytosine. Osmium
oxidation may be effective not only for detection of mC but also for
detection of hmC. Extending the range of hmC detection methods
would be very useful for the next generation of epigenetic studies.
Herein, we report a facile preparation protocol for hmC-
containing oligodeoxynucleotides (ODNs) and their reactivity
Results and discussion
Several routes for the synthesis of hmC-containing ODNs have
been reported.8,9 They include troublesome synthetic steps and/or
many purification processes. We initially developed a facile
synthetic route for hmC-containing ODNs to obtain an efficient
supply of ODNs for the chemical analysis of hmC (Scheme 1).
The synthesis started from thymidine (1). The two hydroxy
groups of 1 were initially protected with acetyl groups. After a
simple extraction process, bromination of the C5 methyl group
was carried out using 2,2¢-azobisisobutyronitrile (AIBN) and N-
bromosuccinimide. The crude product was converted into the
modified thymidine 2 by mixing with 3-hydroxypropionitrile and
subsequent purification with silica gel column chromatography.
The C4 of 2 was activated using phosphorus oxychloride and
1,2,4-triazole, and then aminated with ammonia in dioxane.
After the acetyl groups were completely removed using am-
monia in methanol, the C4 amino group was protected with
a di(n-butyl)formamidine group10,11 to give a 5-hydroxymethyl-
2¢-deoxycytidine derivative 3. The nucleoside 3 was converted
into the phosphoramidite form 4 for the DNA autosynthesizer.
The synthesis of hmC-containing ODN was achieved through the
conventional phosphoramidite method in a DNA autosynthesizer
◦
and subsequent deprotection in 28% ammonia at 70 C for 16 h
(Fig. 1).
hmC-containing ODN formed a stable duplex with the com-
plementary DNA. For example, the melting temperature (Tm) of
the duplex formed by ODN1(hmC) 5¢-CAATGhmCGCTAGT-3¢ and
the complementary ODN ODN1¢(hmC) 5¢-ACTAGhmCGCATTG-
3¢ (1 mM) was 50 ◦C in 50 mM sodium phosphate (pH = 7.0) and
100 mM sodium chloride, which was almost the same ◦value as
that of a methylated duplex ODN1(mC)/ODN1¢(mC) (49 C). The
Tm values of hemimethylated duplexe◦s ODN1(hmC)/ODN1¢(mC)
and ODN1(mC)/ODN1¢(hmC) were 50 C and 49 ◦C, respectively,
aAdvanced Science Institute, RIKEN, Wako, Saitama 351-0198, Japan.
E-mail: aki-okamoto@riken.jp; Fax: +81 48 467 9205; Tel: +81 48 467
9238
bPRESTO, Japan Science and Technology Agency, 4-1-8 Honcho,
Kawaguchi, Saitama 332-0012, Japan
† Electronic supplementary information (ESI) available: Fig. S1 and S2,
and CIF file of the (5R,6S)-hydroxythymidine glycol–dioxidoosmium(VI)–
bipyridine ternary complex. CCDC reference number 814706. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c1ob05247k
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Scheme 1 Facile synthesis of hmC-containing ODN.
The enzymatic digestion of hmC-containing ODNs showed
almost the same result as that of mC-containing ODNs. In
treatment◦with phosphodiesterase and alkaline phosphatase for
2 h at 37 C, the hmC-containing ODN ODN1(hmC) was digested
completely to nucleosides. Treatment of ODN1(hmC)/ODN1¢(hmC)
with the methylation-sensitive restriction enzyme HhaI at
37 ◦C for 1 h showed no cleavage for the methylated du-
plex ODN1(mC)/ODN1¢(mC), while the unmethylated duplex
ODN1(C)/ODN1¢(C) was completely digested.
Osmium oxidation is an excellent method for detection of
5-methylated pyrimidines.7,12,13 Based on this reaction, we have
reported the sequence-selective oxidation of mC.14 The hmC nu-
cleotide may also be reactive on osmium oxidation, although
the effect of addition of a hydroxy group on the reactivity is
unknown. If hmC shows oxidation reactivity as high as that of
mC, hmC detection using osmium oxidation will be possible as with
the detection of mC. The osmium oxidation of hmC-containing
ODNs was tested under the reaction conditions as follows: 5 mM
potassium osmate, 100 mM potassium hexacyanoferrate(III), and
100 mM bipyridine in 100 mM Tris-HCl buffer (pH = 7.7), 1 mM
EDTA, and 10% acetonitrile. The fluorescein-labeled ODN, 5¢-
Fluo-AAAAAAGXGAAAAAA-3¢ (ODN2(X), X = hmC, m◦C, or
C) was added to the reaction mixture and incubated at 0 C for
a period of 5 min. Then, an aliquot of the reaction sample was
treated with hot piperidine to cleave the strands at the reaction
sites. After the reaction, we observed new retarded bands for
ODN2(hmC) and ODN2(mC) in the PAGE analysis (Fig. 2a),
whereas the intensity of the new band was weak for ODN2(C).
After hot piperidine treatment of an aliquot of the reaction
product, these retarded bands disappeared, and instead new
bands appeared in the lanes of ODN2(hmC) and ODN2(mC). The
band mobility, agreeing with that of the short fragment 5¢-Fluo-
AAAAAAGp-3¢ (p = 3¢-phosphate end), indicated that hmC and
mC were selectively oxidized and subsequently cleaved (Fig. 2b).
The reactivity of ODN2(hmC) (61%) was almost the same as that
observed for ODN2(mC) (72%). The reactivity of ODN2(C) was
much lower (28%). The MALDI-TOF MS data of ODN2(hmC)
after oxidation suggested that a stable hydroxymethylcytosine
Fig.
1
ODNs containing 5-hydroxymethylcytosine. (a) hmC- or
mC-containing ODN sequences used in this study. (b) Reverse phase HPLC
profile of synthesized ODN1(hmC). The sample was eluted with a solvent
mixture of 0.1 M triethylammonium acetate (pH = 7.0), linear gradient
over 20 min from 5% to 20% acetonitrile.
suggesting that the addition of one hydroxy group has little
influence on the duplex stability in this case. The CD spectrum
of the duplex ODN1(hmC)/ODN1¢(hmC) exhibits a positive Cotton
effect at 270 nm and a negative Cotton effect at 250 nm, suggesting
that the duplex exhibits a typical B-type DNA structure.
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Fig. 3 The crystal structure of the (5R,6S)-hydroxythymidine glycol—
dioxidoosmium(VI)–bipyridine ternary complex.
We have developed a functional nucleoside in which an adenine
base and a bipyridine ligand were connected with an alkyl
chain. The ODN containing this nucleotide was called the
ICON probe, which is useful for sequence-selective detection
of mC in DNA mediated by osmium complexation. We next
prepared a fluorescein-labeled DNA containing C, mC, or hmC,
ODN2(X), and the ICON probe was designed so that a bipyridine-
labeled adenine base (B) was positioned opposite the target
X base, 5¢-TTTTTTCBCTTTTTT-3¢ (ODN2¢(ICON)) (Fig. 4a).
The reaction mixture was incubated at 25 ◦C for a period of
10 min. Reaction of ODN2(C) was not observed in the mixture
containing oxidants and ODN2¢(ICON). Conversely, the reaction
of ODN2(hmC) in the presence of both potassium osmate and
Fig. 2 Osmium oxidation of mC- and hmC-containing ODN. (a) Forma-
tion of adduct. The fluorescein-labeled ODN2(X) (5 mM) to be examined
was incubated in a solution of 5 mM potassium osmate, 100 mM potassium
hexacyanoferrate(III), 100 mM bipyridine, 0.5 mM EDTA, and 1 M sodium
chloride in 50 mM Tris-HCl buffer (pH 7.7) and 10% acetonitrile at 0 ◦C
for 5 min. (b) Cleavage at the oxidation site by hot piperidine treatment.
The reaction sample was heated in 50 mL of 10% piperidine at 90 ◦C for
20 min. The control DNA was 5¢-AAAAAAGp-3¢.
glycol–dioxidoosmium–bipyridine ternary complex was formed
in the ODN ([M - OH]-, calcd. 5604.99, found 5604.45; [M - 2O -
H]-, calcd. 5588.99, found 5590.14; [M - 3O - H]-, calcd. 5572.99,
found 5575.40) (Scheme 2). The complex formation was supported
by the generation of a retarded band in the PAGE analysis after
osmium oxidation. Another fluorescein-labeled ODN, 5¢-Fluo-
AAAhmCGAGhmCGAAAAAA-3¢ (ODN3(hmC)) also exhibited
osmium oxidation at each hmC (Fig. S1). Osmium oxidation was
observed at hmCs regardless of the number of hmCs in the sequences.
Scheme 2 Osmium oxidation of an hmC-containing ODN.
The structure of the complex was determined by X-ray crystal-
lography of the Os-oxidized product of hydroxythymidine, which
was prepared by the deblocking of 2. The complex obtained from
the osmium oxidation of hydroxythymidine was a hydroxythymi-
dine glycol–dioxidoosmium–bipyridine ternary complex, which
had a slightly distorted octahedral geometry with coplanar glycol
oxygens and bipyridine nitrogens (Fig. 3). The two Os–O double
bonds were trans. The structure of glycol in the complex was
the 5R,6S-configuration. In addition, the hydroxymethyl group
extended from the nucleobase did not coordinate to an osmium
complex core. The N-glycosyl bond was in an anti-conformation,
and the puckering of the ribose ring remained in the C2¢-endo
conformation. The ternary complex structure is close to that
reported for the osmium oxidation of thymidine.15
Fig. 4 Osmium oxidation mediated by the ICON probe. (a) ICON probe
and the interstrand cross-link with hmC-containing ODN. (b) Interstrand
cross-link formation. (c) PAGE analysis after hot piperidine treatment of
the samples shown in (b).
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ODN2¢(ICON) produced a product with a higher molecular
weight, which appeared as a band of low mobility on PAGE
analysis (Fig. 4b). It was similar to the reaction product from
ODN2(mC) and ODN2¢(ICON). Treatment of the reaction product
with hot piperidine eliminated the high-molecular-weight band
and produced a new band that indicated cleavage of the strand at
hmC (Fig. 4c). The appearance of this new band suggests that an in-
terstrand cross-link, mediated by osmium complexation, occurred
between ODN2(hmC) and ODN2¢(ICON). The mass spectrometric
data for the product generated after osmium complexation with
ODN2(hmC)/ODN2¢(ICON) directly indicate the production of a
cross-linked adduct ([M - OH]-, calcd 10238.15, found 10236.40).
The reactivity of ODN2(hmC) in the presence of ODN2¢(ICON)
was 23%, close to that of ODN2(mC) (24%), clearly distinguishable
from that of ODN2(C) (4%). For effective oxidation of hmC,
hybridization of the ICON probe with the target DNA sequence
is essential. For example, a noncomplementary ODN, 5¢-Fluo-
AGCAAhmCGAAGCAAAA-3¢ (ODN4(hmC)), exhibited osmium
oxidation at hmC when the combination of potassium osmate,
potassium hexacyanoferrate(III) and bipyridine was used, whereas
oxidation was not observed at hmC in ODN4(hmC) at all when
ODN2¢(ICON) was used in the presence of potassium osmate and
potassium hexacyanoferrate(III), because ODN2¢(ICON) does not
hybridize with ODN4(hmC) (Fig. S2).
was stirred at room temperature for 2 h. After concentrating,
ethyl acetate (100 mL) and brine (100 mL) were added to the
residue. The product was extracted to the organic layer. The
organic layer was washed with saturated sodium chloride (100 mL)
twice, dried over magnesium sulfate, and filtered. The solvent
was removed by evaporation, and the residue was dried under
reduced pressure. N-Bromosuccinimide (1.96 g, 11 mmol) and
benzene (20 mL) were added to the residue. The mixture was
stirred and repeatedly degassed by reduction of the pressure
and injection of nitrogen gas. After addition of AIBN (164 mg,
1 mmol), the mixture was refluxed for 1 h. The reaction mixture
was cooled and the precipitate was removed by filtration. The
solvent was removed by evaporation and the residue was dried
under reduced pressure. N,N-Dimethylformamide (10 mL) and 3-
hydroxypropionitrile (4.1 mL, 60 mmol) were added to the residue.
The mixture was stirred at room temperature for 24 h. Ethyl acetate
(100 mL) and brine (100 mL) were added to the reaction mixture.
The product was extracted to the organic layer. The organic layer
was washed with brine (100 mL) twice, dried over magnesium
sulfate, and filtered. The solvent in the organic phase was removed
by evaporation. The product was purified by silica gel column
chromatography (2 : 1–1 : 0 ethyl acetate/hexane). The product 2
was obtained as a white foam (1.80 g, 46%): Rf 0.48 (ethyl acetate);
1H NMR (CDCl3) d 9.33 (br, 1H), 7.63 (t, J = 1.0, 1H), 6.31 (dd,
J = 8.4, 5.5, 1H), 5.24–5.22 (m, 1H), 4.41–4.27 (m, 5H), 3.80–
3.76 (m, 2H), 2.52 (ddd, J = 14.2, 5.6, 1.7, 1H), 2.24 (ddd, J =
14.2, 8.3, 6.4, 1H), 2.13 (s, 3H), 2.12 (s, 3H); 13C NMR (CDCl3) d
170.24, 170.23, 162.7, 150.1, 137.4, 117.8, 111.4, 85.3, 82.3, 74.2,
65.3, 64.9, 63.7, 37.4, 20.72, 20.65, 18.59; HRMS (ESI) calcd
for C17H21N3O8Na ([M + Na]+) 418.1221, found 418.1221; UV
(chloroform) lmax 264 nm (e 34260). Thymidine diacetate (719 mg,
22%) was also collected: Rf 0.63 (ethyl acetate).
Conclusions
In conclusion, we synthesized ODN strands containing hmC and
investigated their reactivity on osmium oxidation. The osmium
oxidation of hmC-containing ODN provided a 5-hydroxymethyl-2¢-
deoxycytidine glycol–dioxidoosmium–bipyridine ternary complex
in the ODN. Characterization of the structure of the osmium-
centered complex in the ODN would support new drug design
for efficient DNA hydroxymethylation analysis. Although there
still remain further aspects to be examined toward an easier-to-
use hydroxymethylation analysis, such as distinguishing between
hmC and mC, we anticipate that this new concept of effective
hydroxymethylation analysis supported by the chemical basis will
be the starting point for an epoch-making epigenotyping assay,
which will supersede conventional methods.
4-N,N-Di-n-butylformamidine-5-(2-cyanoethoxy)methyl-2¢-
deoxycytidine (3)
Nucleoside 2 (1.80 g, 4.55 mmol) was dissolved in acetonitrile
(80 mL). Ethyl acetate (6.98 mL, 50.1 mmol) and phosphorus
oxychloride (0.85 mL, 9.1 mmol) were added to the solution with
stirring. The reaction mixture was stirred at room temperature
for 1 h. The solvent was evaporated, and ethyl acetate (100 mL)
and brine (100 mL) were added to the residue. The product was
extracted to the organic layer. The organic layer was washed with
brine twice and dried over magnesium sulfate. The solvent was
removed by evaporation, and the residue was dried under reduced
pressure. 1,4-Dioxane (100 mL) and 28% ammonia (15 mL) were
added to the residue. The mixture was stirred at room temperature
for 1 h. The solvent was removed by evaporation. Methanol
(15 mL) and 28% ammonia (15 mL) were added to the residue. The
mixture was stirred at room temperature for 4 h. The solvent was
removed by evaporation, and then the residue was azeotropically
dried with methanol under reduced pressure. Methanol (20 mL)
and N,N-di-n-butylformamide dimethyl acetal (4.64 g, 22.8 mmol)
were added to the residue. The mixture was stirred at room
temperature for 1 h. The solvent was removed by evaporation.
The product was purified by silica gel column chromatography
(0–10% methanol/ethyl acetate). The product 3 was obtained as
a white solid (1.41 g, 69%): mp 67 ◦C; 1H NMR (CD3OD) d 8.64
(s, 1H), 8.21 (s, 1H), 6.27 (t, J = 6.4, 1H), 4.50–4.44 (m, 2H), 4.37
Experimental
General
1H, 13C, and 31P NMR spectra were measured with a Varian
NMR system 500. Coupling constants (J value) are reported
in hertz. The chemical shifts are shown in ppm downfield from
tetramethylsilane, using residual chloroform (d = 7.27 in 1H NMR,
d = 77.0 in 13C NMR) and methanol (d = 3.30 in 1H NMR, d = 49.0
in 13C NMR) as an internal standard. An external H3PO4 standard
(d = 0.00 ppm) was used for 31P NMR measurements. ESI mass
spectra were recorded on a Bruker BioApex II. MALDI-TOF
mass spectra were measured with a Bruker Daltonics Reflex.
3¢O,5¢O¢-Diacetyl-5-(2-cyanoethoxy)methyl-2¢-deoxyuridine (2)
Thymidine (2.42 g, 10 mmol) was suspended in acetonitrile
(20 mL). Triethylamine (8.4 mL, 60 mmol) and acetic anhydride
(3.8 mL, 40 mmol) were added to the suspension. The mixture
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(ddd, J = 6.4, 3.9, 3.9, 1H), 3.96 (m, 1H), 3.82 (dd, J = 12.1, 3.3
Hz, 1H), 3.75 (dd, J = 12.0, 3.9, 1H), 3.71 (t, J = 6.1, 2H), 3.60 (t,
J = 7.6, 2H), 3.45 (t, J = 7.3, 2H), 2.71 (t, J = 6.0, 2H), 2.41 (ddd,
J = 13.6, 6.2, 4.0, 1H), 2.17 (ddd, J = 13.4, 6.7, 6.7, 1H), 1.69–
1.62 (m, 4H), 1.42–1.32 (m, 4H), 0.99–0.95 (m, 6H); 13C NMR
(CD3OD) d 171.5, 159.1, 158.3, 142.4, 119.7, 112.7, 89.0, 88.0,
71.9, 67.6, 66.3, 62.7, 53.4, 47.1, 42.3, 32.0, 30.2, 21.2, 20.7, 19.3,
14.2, 14.0; HRMS (ESI) calcd for C22H35N5O5Na ([M + Na]+)
472.2530, found 472.2530; UV (methanol) lmax 325 nm (e 45690),
233 nm (e 42980).
calcd 3676.4, found 3676.3; ODN2(hmC), [M + H]+, calcd 5212.6,
found 5211.5; ODN2¢(ICON), [M + H]+, calcd 4790.3, found
4790.9.
Osmium oxidation (bipyridine use)
The fluorescein-labeled ODN2(X) (5 mM) to be examined was
incubated in a solution of 5 mM potassium osmate, 100 mM
potassium hexacyanoferrate(III), 100 mM bipyridine, 0.5 mM
EDTA, and 1 M sodium chloride in 50 mM Tris-HCl buffer
(pH 7.7) and 10% acetonitrile at 0 ◦C for 5 min. The reaction
solution was filtered to deionize it, and then the sample was
precipitated in ethanol. After drying in vacuo, the precipitated
DNA◦was redissolved in 50 mL of 10% piperidine (v/v), heated
at 90 C for 20 min, and then evaporated to dryness by vacuum
rotary evaporation.
Phosphoramidite (4)
Nucleoside 3 (1.41 g, 3.14 mmol) and 4,4¢-dimethoxytrityl chloride
(1.38 g, 4.08 mmol) were dissolved in pyridine, and the solution was
stirred at room temperature for 2 h. Methanol (1 mL) was added to
the reaction mixture, and the solvent was evaporated. Pyridine in
the residue was removed by coevaporation with dichloromethane
and hexane. The product was purified by silica gel column chro-
matography (1% triethylamine, 0–2% methanol/ethyl acetate).
The product (1.46 g, 62%) was obtained as a white foam: mp 70 ◦C;
1H NMR (CDCl3) d 8.79 (s, 1H), 8.07 (s, 1H), 7.46–7.19 (m, 9H),
6.84–6.82 (m, 4H), 6.47 (t, J = 6.5, 1H), 4.58 (br, 1H), 4.52 (br, 1H),
4.19–4.15 (m, 2H), 3.96 (d, J = 11.5, 1H), 3.77 (s, 6H), 3.55–3.47
(m, 3H), 3.35–3.29 (m, 5H), 2.69 (ddd, J = 13.5, 5.6, 3.2, 1H), 2.22
(ddd, J = 13.6, 6.7, 6.7, 1H), 2.09 (t, J = 6.8, 2H), 1.64–1.56 (m, 4H),
1.38–1.24 (m, 4H), 0.96–0.92 (m, 6H); 13C NMR (CDCl3) d 169.6,
158.2, 158.2, 157.5, 156.1, 144.4, 140.6, 135.5, 135.4, 129.9, 129.8,
127.9, 127.6, 126.6, 117.4, 112.9, 110.5, 86.1, 86.0, 85.8, 71.4, 66.1,
64.5, 63.2, 54.9, 52.1, 45.5, 42.0, 30.7, 28.7, 19.9, 19.5, 17.7, 13.5,
13.4; HRMS (ESI) calcd for C43H54N5O7 ([M + H]+) 752.4018,
found 752.4019; UV (chloroform) lmax 324 nm (e 32010), 242 nm
(e 23590). The tritylated nucleoside (270 mg, 0.36 mmol) and 1H-
tetrazole (50 mg, 0.72 mmol) were dissolved in acetonitrile (5 mL),
and 2-cyanoethyl N,N,N¢,N¢-tetraisopropyl phosphorodiamidite
(343 mL, 1.08 mmol) was added to the solution. The reaction
mixture was stirred at room temperature for 1 h. A mixture of
ethyl acetate (25 mL) and saturated sodium bicarbonate (25 mL)
was added to the reaction mixture. The product was extracted
to the organic layer. The organic layer was washed with brine
twice and dried over magnesium sulfate. The solvent was removed
by evaporation. The product was dried by coevaporation with
acetonitrile and further dried under reduced pressure. 31P NMR
(CDCl3): d 149.660, 149.028; HRMS (ESI) calcd for C52H71N7O8P
([M + H]+) 952.5096, found 952.5099. Acetonitrile (2.4 mL) was
added to the residue, and the solution was passed through a
0.45 mm filter. The resulting solution was used for automated DNA
synthesis without further purification.
Osmium oxidation (ICON DNA use)
The fluorescein-labeled ODN2(X) (8 mM) to be examined was
incubated in a solution of 5 mM ODN2¢(ICON), 5 mM potassium
osmate, 100 mM potassium hexacyanoferrate(III), 0.5 mM EDTA,
and 1 M sodium chloride in 50 mM Tris-HCl buffer (pH 7.7) at
◦
25 C for 10 min. The reaction solution was filtered to deionize
it, and then the sample was precipitated in ethanol. After drying
in vacuo, the precipitated DNA was redissolved in 50 mL of 10%
piperidine (v/v), heated at 90 ◦C for 20 min, and then evaporated
to dryness by vacuum rotary evaporation.
Melting temperature (Tm) measurements
All Tm measurements of the DNA duplexes (1 mM, final duplex
concentration) were made in 50 mM sodium phosphate buffer
(pH 7.0) containing 100 mM sodium chloride. Absorbance vs.
temperature profiles were measured at 260 nm with a Shimadzu
UV-2550 spectrophotometer equipped with a Peltier temperature
controller using a cell with a 1 cm path length. The absorbance of
◦
◦
the samples was ◦monitored at 260 nm from 10 C to 90 C, at a
heating rate of 1 C min-1.
Acknowledgements
We thank Dr Takemichi Nakamura and Dr Kazuki Tainaka
(RIKEN) for the mass spectrometry. We also thank Dr Daisuke
Hashizume (RIKEN) for the X-ray structural analysis.
Notes and references
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DNA synthesis and characterization
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4180 | Org. Biomol. Chem., 2011, 9, 4176–4181
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Org. Biomol. Chem., 2011, 9, 4176–4181 | 4181
©