Site-directed spin-labeling of nucleic acids by click chemistry: Detection of abasic sites in duplex DNA by EPR spectroscopy

Article abstract of DOI:10.1021/ja102797k

This paper describes a spin label that can detect and identify local structural deformations in duplex DNA, in particular abasic sites. The spin label was incorporated into DNA by a new postsynthetic approach using click-chemistry on a solid support, which simplified both the synthesis and purification of the spin-labeled oligonucleotides. A nitroxide-functionalized azide, prepared by a short synthetic route, was reacted with an oligomer containing 5-ethynyl-2′-dU. The conjugation proceeded in quantitative yield and resulted in a fairly rigid linker between the modified nucleotide and the nitroxide spin label. The spin label was used to detect, for the first time, abasic sites in duplex DNA by X-band CW-EPR spectroscopy and give information about other structural deformations as well as local conformational changes in DNA. For example, reduced mobility of the spin label in a mismatched pair with T was consistent with the spin label displacing the T from the duplex. Addition of mercury(II) to this mispair resulted in a substantial increase in the motion of the spin label, consistent with formation of a metallopair between the T and the spin-labeled base that results in movement of the spin label out of the duplex and toward the solution. Thus, reposition of the spin label, when acting as a mercury(II)-controlled mechanical lever, can be readily detected by EPR spectroscopy. The ease of incorporation and properties of the new spin label make it attractive for EPR studies of nucleic acids and other macromolecules.

Full text of DOI:10.1021/ja102797k

Published on Web 07/09/2010
Site-Directed Spin-Labeling of Nucleic Acids by Click
Chemistry: Detection of Abasic Sites in Duplex DNA by EPR
Spectroscopy
Ulla Jakobsen,† Sandip A. Shelke,‡ Stefan Vogel,*,† and Snorri Th. Sigurdsson*,‡
Department of Physics and Chemistry, UniVersity of Southern Denmark, Nucleic Acid Center
CampusVej 55, 5230 Odense, Denmark, and UniVersity of Iceland, Science Institute,
Dunhaga 3, 107 ReykjaVik, Iceland
Received April 2, 2010; E-mail: snv@ifk.sdu.dk; snorrisi@hi.is
Abstract: This paper describes a spin label that can detect and identify local structural deformations in
duplex DNA, in particular abasic sites. The spin label was incorporated into DNA by a new postsynthetic
approach using click-chemistry on a solid support, which simplified both the synthesis and purification of
the spin-labeled oligonucleotides. A nitroxide-functionalized azide, prepared by a short synthetic route,
was reacted with an oligomer containing 5-ethynyl-2′-dU. The conjugation proceeded in quantitative yield
and resulted in a fairly rigid linker between the modified nucleotide and the nitroxide spin label. The spin
label was used to detect, for the first time, abasic sites in duplex DNA by X-band CW-EPR spectroscopy
and give information about other structural deformations as well as local conformational changes in DNA.
For example, reduced mobility of the spin label in a mismatched pair with T was consistent with the spin
label displacing the T from the duplex. Addition of mercury(II) to this mispair resulted in a substantial increase
in the motion of the spin label, consistent with formation of a metallopair between the T and the spin-
labeled base that results in movement of the spin label out of the duplex and toward the solution. Thus,
reposition of the spin label, when acting as a mercury(II)-controlled mechanical lever, can be readily detected
by EPR spectroscopy. The ease of incorporation and properties of the new spin label make it attractive for
EPR studies of nucleic acids and other macromolecules.
the oligonucleotide synthesis (postsynthetic funtionalization).4
The postsynthetic strategy is often advantageous, as synthesis
Introduction
EPR spectroscopy is a valuable technique to study the
structural properties and dynamics of macromolecules, including
oligonucleotides.1 However, these studies require the incorpora-
tion of a spin label (a stable free radical, e.g., a nitroxide) into
the oligonucleotide. This can be accomplished either by direct
incorporation of the corresponding spin label phosphoramidite
during automated synthesis of the oligonucleotide,2 by enzymatic
incorporation of spin-labeled nucleoside triphosphates,3 or by
incorporating a modified nucleotide or another building block
which can selectively react with a spin-labeling reagent after
of spin-labeled phosphoramidites is often tedious and the spin
label may decompose under the conditions employed in
automated DNA synthesis.2a,d Examples of postsynthetic label-
ing include reaction at the amino group in 2′-amino-modified
RNA,4a-c at modified internucleotide linkages,4d,e and at
4-thiouridine.4f,g
The CuI-catalyzed Huisgen-Meldal-Sharpless [3 + 2]
cycloaddition5 (click chemistry)6 between an alkyne and an azide
has been used for modification of nucleobases7 as well as for
(4) (a) Edwards, T. E.; Okonogi, T. M.; Robinson, B. H.; Sigurdsson,
S. T. J. Am. Chem. Soc. 2001, 123, 1527–1528. (b) Kim, N.-K.; Murali,
A.; DeRose, V. J. Chem. Biol. 2004, 11, 939–948. (c) Edwards, T. E.;
Okonogi, T. M.; Sigurdsson, S. T. Chem. Biol. 2002, 9, 699–706. (d)
Nagahara, S.; Murakami, A.; Makino, K. Nucleosides Nucleotides
1992, 11, 889–901. (e) Qin, P. Z.; Butcher, S. E.; Feigon, J.; Hubbell,
W. L. Biochemistry 2001, 40, 6929–6936. (f) Qin, P. Z.; Hideg, K.;
Feigon, J.; Hubbell, W. L. Biochemistry 2003, 42, 6772–6783. (g)
Ramos, A.; Varani, G. J. Am. Chem. Soc. 1998, 120, 10992–10993.
(5) (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem. 2002, 114, 4812-4825; Angew. Chem., Int. Ed. 2002,
41, 2596-2599. (b) Tornøe, C. W.; Christensen, C.; Meldal, M. J.
Org. Chem. 2002, 67, 3057–3064.
† University of Southern Denmark.
‡ University of Iceland.
(1) (a) Qin, P. Z.; Dieckmann, T. Curr. Opin. Struc. Biol. 2004, 14, 350–
359. (b) Schiemann, O.; Prisner, T. F. Q. ReV. Biophys. 2007, 40,
1–53. (c) Sowa, G. Z.; Qin, P. Z. Prog. Nucleic Acid Res. 2008, 82,
147–197. (d) Zhang, X.; Cekan, P.; Sigurdsson, S. T.; Qin, P. Methods
Enzymol. 2009, 469, 303–328.
(2) (a) Gannet, P. M.; Darian, E.; Powell, J.; Johnson, E. M.; Mundoma,
C.; Greenbaum, N. L.; Ramsey, C. M.; Dalal, N. S.; Budil, D. E.
Nucleic Acids Res. 2002, 30, 5328–5337. (b) Spaltenstein, A.;
Robinson, B. H.; Hopkins, P. B. J. Am. Chem. Soc. 1988, 110, 1299–
1301. (c) Barhate, N.; Cekan, P.; Massey, A. P.; Sigurdsson, S. T.
Angew. Chem. 2007, 119, 2709-2712; Angew. Chem., Int. Ed. 2007,
46, 2655-2658. (d) Cekan, P.; Smith, A. L.; Barhate, N.; Robinson,
B. H.; Sigurdsson, S. T. Nucleic Acids Res. 2008, 36, 5946–5954.
(3) Obeid, S.; Yulikov, M.; Jeschke, G.; Marx, A. Angew. Chem. 2008,
120, 6886-6890; Angew. Chem., Int. Ed. 2008, 47, 6782-6785.
(6) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. 2001, 113,
2056-2075; Angew. Chem., Int. Ed. 2001, 40, 2004-2021.
(7) (a) Koe`olka, P.; Andersen, N. K.; Jensen, F.; Nielsen, P. ChemBioChem
2007, 8, 2106–2116. (b) Seela, F.; Sirivolu, V. R. HelV. Chim. Acta
2007, 90, 535–552. (c) Park, S. M.; Shen, Y.; Kim, B. H. Org. Biomol.
Chem. 2007, 5, 610–612.
9
10424 J. AM. CHEM. SOC. 2010, 132, 10424–10428
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Site-Directed Spin-Labeling of Nucleic Acids by Click Chemistry
A R T I C L E S
Scheme 1. Synthesis of Azide-Containing Nitroxide 3
Scheme 2. Synthesis of Nitroxide-Labeled Nucleoside 5 (a) and
Oligonucleotide 7 (b) by Click Chemistrya
the labeling of DNA in solution7b,8 and on solid support.7b The
reaction is attractive, since it can be performed under mild
conditions, tolerates a broad range of functional groups and
reaction conditions, usually gives high yields, and is orthogonal
to most organic reactions and consequently chemoselective.
Here, we report site-directed spin-labeling of DNA by a click
reaction and the use of the new label to detect local structural
deformations in duplex DNA, in particular abasic sites.
Results and Discussion
Synthesis of Spin Label and Postsynthetic DNA Labeling
by Click Chemistry. Since azides are sensitive to the conditions
used in automated DNA synthesis,9 the nitroxide was function-
alized with an azide, rather than the DNA. Thus, the azide-
containing nitroxide 3 was synthesized from the known ami-
noisoindoline 110 by reaction with triflyl azide11 to give azide
2, which was oxidized with mCPBA (Scheme 1).
The alkyne 5-ethynyl-2′-dU 4 was chosen for reaction with
azide 3 as it has previously been used in CuI-catalyzed Huisgen
cycloadditions7a,c and results in a relatively short and rigid linker
between the spin-label and the oligonucleotide. The reaction is
usually performed using a copper(II) salt and sodium ascorbate
as a reducing agent,5a but since these conditions cause degrada-
tion of DNA12 and reduction of nitroxides,13 a copper(I) salt
was used directly. Thus, the commercially available 5-ethynyl-
2′-deoxyuridine 4 was reacted with the azide functionalized
nitroxide 3 in a CuI-catalyzed Huisgen-Meldal-Sharpless [3
+ 2] cycloaddition to yield the spin-labeled uridine derivative
5 (Scheme 2a). The nitroxide functional group was stable under
these reaction conditions.
The 5′-DMT protected phosphoramidite of 5-ethynyl-2′-
deoxyuridine is commercially available, and thus, the alkyne
can easily be incorporated into an oligonucleotide using standard
conditions for automated DNA synthesis. However, due to low
coupling yields obtained with the standard activator 1H-tetrazole,
1.8 M pyridinium hydrochloride was used as an activator and
the coupling time increased to 15 min for the modified amidite.
The optimized coupling conditions resulted in nearly quantitative
coupling yields (DNA sequence shown in Figure 2a).
a The modification of the oligomer takes place before deprotection and
cleavage from the resin. TBTA is the CuI-stabilizing ligand tris-(benzyltriazo-
lylmethyl)amine.
Copper(I)-catalyzed [3 + 2] cycloadditions on oligonucle-
otides containing the 5-ethynyl-2′-deoxyuridine derivative have
been reported in solution with a number of different azides.8a
However, this method8a proved unsuccessful in our hands, and
analysis of the reaction mixture by MALDI-TOF showed only
a minor trace of the spin-labeled oligonucleotide. Prolonged
reaction times resulted in decomposition of the oligonucleotide,
even in the presence of the CuI-stabilizing ligand tris-(benzyl-
triazolylmethyl)amine (TBTA)14 which has been shown to
protect the metal from oxidation and to protect DNA from
degradation by reactions mediated by CuI.8a The oligonucleotide
was consequently reacted with an excess of compound 3, CuBr,
and TBTA while still attached to the solid support (Scheme
2b). After the reaction, the excess reagents were removed by
washing and the oligonucleotide was cleaved from the solid
support using standard conditions (conc. aqueous NH3 for 12 h,
55 °C). Analysis of the reaction mixture by MALDI-TOF
showed a nearly quantitative conversion to the spin-labeled
oligonucleotide (MW 4494.9 g mol-1) (Figure 1). The effect
of the spin label on the stability of duplex DNA was determined
by thermal denaturation experiments. The modification de-
creased the stability of the corresponding DNA duplex by 3.5
°C, when compared to an oligonucleotide containing T instead
of the modified dU derivative (Supporting Information).
Engels and co-workers have also reported an on-column spin-
labeling method using a Sonogashira coupling.15a However, after
spin-labeling, the oligomer synthesis is continued, which exposes
the spin label to the nucleic acid synthesis reagents. This causes
a partial reduction of the nitroxides.15b In contrast, the on-column
method described here is a postsynthetic approach, that is, the
(8) (a) Gierlich, J.; Burley, G. A.; Philipp, M. E. G.; Hammond, D. E.;
Carell, T. Org. Lett. 2006, 8, 3639–3642. (b) Burley, G. A.; Gierlich,
J.; Mofid, M. R.; Nir, H.; Tal, S.; Eichem, Y.; Carell, T. J. Am. Chem.
Soc. 2006, 128, 1398–1399.
(9) Lietard, J.; Meyer, A.; Vasseur, J.-J.; Morvan, F. Tetrahedron Lett.
2007, 48, 8795–8798.
(10) Miller, T. R.; Hopkins, P. B. Bioorg. Med. Chem. Lett. 1994, 4, 981–
986.
(11) Liu, Q.; Tor, Y. Org. Lett. 2003, 5, 2571–2572.
(12) (a) Chiou, S.-H. J. Biochem. 1983, 94, 1259–1267. Oikawa, S.;
Kawanishi, S. Biochemistry 1996, 35, 4584–4590. (b) Kumar, R.; El-
Sagheer, A.; Tumpane, J.; Lincoln, P.; Wilhelmsson, L. M.; Brown,
T. J. Am. Chem. Soc. 2007, 129, 6859–6864.
(14) Lee, B. Y.; Park, S. R.; Jeon, H. B.; Kim, K. S. Tetrahedron Lett.
2006, 47, 5105–5109.
(15) (a) Schiemann, O.; Piton, N.; Plackmeyer, J.; Bode, B. E.; Prisner,
T. F.; Engels, J. W. Nat. Protoc. 2007, 2 (4), 904–923. (b) Piton, N.;
Mu, Y. G.; Stock, G.; Prisner, T. F.; Schiemann, O.; Engels, J. W.
Nucleic Acids Res. 2007, 35, 3128–3143.
(13) Yu, Z.; Kotake, Y.; Janzen, E. G. Redox Rep. 1996, 2, 133–139.
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Jakobsen et al.
Figure 1. MALDI-TOF spectrum of the crude click reaction mixture
between azide 3 and 5-ethynyl-2′-dU modified oligonucleotide 6 (MW )
4263.7 g mol-1) to yield spin-labeled oligonucleotide 7 (MW ) 4494.9 g
mol-1). The response of the alkyne-containing oligomer in the MALDI-
TOF was shown in a separate experiment to be within a maximal factor of
2 of the spin-labeled oligomer.
oligomer has already been synthesized when the spin labeling
is carried out, and therefore, the spin label is not exposed to
harmful reagents. Another advantage of this on-column protocol
is that it is not moisture sensitive.
Figure 2. EPR spectra of the spin-labeled nucleoside (5), ssDNA, and
DNA duplexes opposite an A or an abasic site (F). (a) Sequences of the
spin-labeled duplexes. The identity of each spectrum is shown in insets.
(b) EPR spectra of spin-labeled DNAs and the free spin label 5 at 30 °C.
5 is only soluble in DMSO and was measured at 30 °C because of the
relatively high freezing point of DMSO. (c) EPR spectra of the fully matched
duplex and a duplex containing the abasic site opposite the spin label
measured at 10 °C.
EPR Studies. Incorporation of the nitroxide into DNA was
verified by continuous-wave (CW) EPR spectroscopy, which
showed the expected three-line pattern for a nitroxide (Figure
2). EPR spectra of nitroxide radicals also give information about
their mobility; as the motion slows down, the spectrum becomes
wider. Thus, the EPR spectrum of the spin-labeled nucleoside
5, which can tumble freely in solution, has narrower lines than
the spin-labeled single stranded DNA (Figure 2b). As expected,
the spectral width increased even more after annealing the spin-
labeled single strand to the complementary oligomer (see Figure
S1 in Supporting Information for comparison of ssDNA and
duplex DNA at the same temperature). However, a slow-moving
component of the single strand spectrum indicates a minor
foldamer where the spin label has lower mobility than in the
duplex.
removal of damaged nucleic bases by DNA glycosylases.16
These lesions have been detected by a number of different ways,
including fluorescent probes, either incorporated into DNA17
or bound to specific sites in DNA,18 mass spectrometry,19
acoustic wave,20 and electrochemical methods.20b,21 Pulsed EPR
spectroscopy of doubly spin-labeled duplex DNA in frozen
solutions has been used to measure a shortening of DNA helices
containing an abasic site,22 but until now, CW EPR spectroscopy
has not been used to directly detect an abasic site.
However, the EPR spectrum of the spin-labeled duplex DNA
is considerably narrower than what has been observed for the
rigid nitroxide C¸ in duplex DNA.2d Thus, there is some motion
in the linker connecting the nitroxide to the nucleoside. The
motion of the spin label, independent of the duplex, results from
rotation of the two single bonds that connect the triazole to the
base and the nitroxide moiety. Since all the atoms in the tether
are sp2-hybridized, the three aromatic rings can lie in the same
plane, as supported by modeling studies of a triazole ring
connected at the 5-position of a pyrimidine nucleobase.7a
Furthermore, if rotation around the single bonds connecting the
rings could be restricted, the motion of the nitroxide would be
reduced and result in a much broader EPR spectrum. Thus, EPR
spectroscopy could be used to detect sites to which the extended
aromatic system of the label could bind and thereby substantially
slow the motion of the nitroxide. We have successfully used
this promising feature of the spin label to detect an abasic site
in DNA by EPR spectroscopy.
The single-stranded, spin-labeled oligomer was annealed to
a DNA strand containing a stable analogue of an abasic site,
3-hydroxy-2-(hydroxymethyl)-tetrahydrofuran. Introduction of
an abasic site creates a cleft into which the spin-label can
(16) (a) Lindahl, T. Nature 1993, 362, 709–715. (b) Lhomme, J.; Constant,
J.-F.; Demeunynck, M. Biopolymers 1999, 52, 65–83.
(17) (a) Greco, N. J.; Tor, Y. J. Am. Chem. Soc. 2005, 127, 10784–10785.
(b) Valis, L.; Amann, N.; Wagenknecht, H.-A. Org. Biomol. Chem.
2005, 3, 36–38.
(18) Rajendar, B.; Nishizawa, S.; Teramae, N. Org. Biomol. Chem. 2008,
6, 670–673.
(19) Zhang, L.-K.; Gross, M. L. J. Am. Soc. Mass Spectrom. 2002, 13,
1418–1426.
(20) (a) Hianik, T.; Wang, X.; Andreev, S.; Dolinnaya, N.; Oretskaya, T.;
Thompson, M. Analyst 2006, 131, 1161–1166. (b) Bartosˇ´ık, M.;
Gajdosˇ, V.; Kostee`ka, P.; Fojta, M.; Palee`ek, E.; Volkov, E.;
Ooretskaya, T.; Hianik, T. Electroanalysis 2009, 21, 295–302.
(21) (a) Dolinnaya, N. G.; Jan, M. R.; Kawde, A. N.; Oretskaya, T. S.;
Tashlitsky, V. N. Electroanalysis 2006, 4, 399–404. (b) Buzzeo, M. C.;
Barton, J. K. Bioconjugate Chem. 2008, 19, 2110–2112.
(22) Sicoli, G.; Mathis, G.; Aci-Seche, S.; Saint-Pierre, C.; Boulard, Y.;
Gasparutto, D.; Gambarelli, S. Nucleic Acids Res. 2009, 37, 3165–
3176.
Abasic sites are common lesions that result from spontaneous
hydrolytic cleavage of the N-glycosidic bond in DNA or from
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Site-Directed Spin-Labeling of Nucleic Acids by Click Chemistry
A R T I C L E S
Figure 3. Putative model for the location of the spin label (yellow) when intercalated in the duplex cleft formed by an abasic site. Side view (left) and top
view (right) with π-π interactions indicated by orange lines. H-atoms have been omitted for clarity.
intercalate, driven by favorable stacking interactions with
neighboring bases as well as hydrophobicity of the label. Indeed,
the EPR spectrum of the DNA duplex containing a spin label
directly across from the abasic site shows significant broadening
(Figure 2c). This is consistent with the spin label flipping into
the abasic site pocket with concomitant reduction of its motion,
in contrast to the fully base-paired duplex where the spin label
has more degrees of freedom as it extends into the solution. A
model of the spin label (Figure 3) shows that the spin label fits
well in the abasic site, where it forms a network of favorable
π-π interactions while placing the methyl groups of the
tetramethylindoline nitroxide moiety into the major groove. The
spin-labeled base is also stacked within the duplex, which is
consistent with NMR studies that have shown that a T in a
similar sequence context placed opposite an abasic site stacks
within the helix.23 In addition to the slow motion component
in the EPR spectrum, there are also narrower lines that indicate
a faster moving component with mobility similar to that obtained
for the single strand/duplex. Double integration of the EPR
spectrum shows that ca. 80% of the sample contains the spin
label in the abasic site (Supporting Information).
How specific is the detection of an abasic site by 7? Do other
structural deformations in the vicinity of 7 show a similar EPR
spectrum? To determine if the new spin label could be used to
differentiate abasic sites from other structural lesions by EPR
spectroscopy, DNA duplexes with different mismatches opposite
the spin label were prepared (Figure 4a) and their EPR spectra
recorded (Figure 4b). The spectra of the individual mismatches
are different from each other, although the difference is not as
clear as when a TEMPO-spin-labeled dC was used to detect
and distinguish between different bases.24 For example, the EPR
spectra of 7:A and 7:C are quite similar. On the other hand,
both 7:T and 7:G show a slow-moving component that can be
used to distinguish each pair from all others. The only pair that
shows a somewhat similar spectrum to the abasic site is 7:T;
however, the two spectra are clearly distinguishable (Figure 4c).
The fact that 7:T, shows a slow-moving component indicates
that the spin label is displacing the opposing T and stacking
within the duplex. If that is the case, it should be possible to
change the mobility of the spin label by adding Hg2+, which
can stabilize T:T25 and U:U mismatches26 by bridging the N3
Figure 4. EPR spectra of DNA duplexes that contain spin label 7 opposite
different bases (A, T, G, C) or an abasic site (F) at 10 °C. (a) Sequences
of the spin-labeled duplexes. (b-d) EPR spectra of spin-labeled DNAs.
The identity of each spectrum is shown in insets.
atoms of the nucleobases.27 In fact, formation of a 7-Hg-T base
pair upon addition of Hg2+ should place the spin label in a
similar position as in the fully matched duplex and thereby
dramatically increase the mobility of the nitroxide. Indeed, the
(23) Coppel, Y.; Berthet, N.; Coulombeau, C.; Coulombeau, C.; Garcia,
J.; Lhomme, J. Biochemistry 1997, 36, 4817–4830.
(24) Cekan, P.; Sigurdsson, S. T. J. Am. Chem. Soc. 2009, 131, 18054–
18056.
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spectra of 7:A and 7:T in the presence of Hg2+ are nearly
superimposable (Figure 4d). Thus, mercuric ions can effect
displacement of the spin label out of the duplex and into the groove
in a controlled fashion. Such a mercuric switch might find use for
mechanical maneuvering of nucleic acid nanostructures.
is an excellent probe for detecting abasic sites in duplex DNA
by X-band CW-EPR spectroscopy. An advantage of using EPR
for detection of abasic sites, compared to other methods, is that
it is based on the mobility of the spin label. In contrast,
fluorescence-based methods detect changes in fluorescence that
can be influenced by factors other than the abasic site, such as
identity of the flanking bases. Another advantage of EPR over
fluorescence is the lack of background interference when doing
measurements in a biological matrix. The spin label described
here can also give information about local structural lesions,
other than abasic sites. For example, reduced mobility of the
spin label in a 7:T mismatch in a duplex DNA was consistent
with the spin label displacing the T. Addition of mercury(II)
resulted in an increased motion of the spin label, consistent with
formation of a 7-Hg-T metallopair that flips the spin label out
toward the solution. The azide-functionalized nitroxide 3 can
also be used to spin-label other macromolecules using click
chemistry.
Conclusion
In conclusion, we have incorporated a new spin label in a
DNA strand by a new postsynthetic method using click-
chemistry on a solid support, simplifying both the synthesis and
purification of the spin-labeled oligonucleotides. The azide-
functionalized nitroxide used in the reaction was synthesized
by a short synthetic route and reacted with oligomers containing
5-ethynyl-2′-dU, the phosphoramidite of which is commercially
available. The incorporation proceeds in excellent yield, avoids
possible break down of the spin-label during oligonucleotide
synthesis, and results in a fairly rigid linker between the modified
nucleotide and the nitroxide spin label. This particular spin label
Acknowledgment. This work was supported by the Danish
National Research Foundation, the Icelandic Research Fund
(080041022), and a doctoral fellowship to S.A.S from the University
of Iceland.
(25) (a) Torigoe, H.; Kawahashi, K.; Takamori, A.; Ono, A. Nucleosides,
Nucleotides Nucleic Acids 2005, 24, 915–917. (b) Miyake, Y.; Togashi,
H.; Tashiro, M.; Yamaguchi, H.; Oda, S.; Kudo, M.; Tanaka, Y.;
Kondo, Y.; Sawa, R.; Fujimoto, T.; Machinami, T.; Ono, A. J. Am.
Chem. Soc. 2006, 128, 2172–2173. (c) Ono, A.; Togashi, H. Angew.
Chem. 2004, 116, 4400-4402; Angew. Chem., Int. Ed. 2004, 43,
4300-4302.
Supporting Information Available: Experimental methods,
including synthetic procedures, Tm studies, and EPR spectros-
copy. This material is available free of charge via the Internet
at http://pubs.acs.org.
(26) Kozasa, T.; Miyakawa, Y.; Ono, A.; Torigoe, H. Nucleic Acids Symp.
Ser. 2008, 52, 197–198.
(27) (a) Tanaka, Y.; Oda, S.; Yamaguchi, H.; Kudo, M.; Kondo, Y.; Kijima,
C.; Ono, A. Nucleic Acids Symp. Ser. 2006, 50, 47–48. (b) Tanaka,
Y.; Oda, S.; Yamaguchi, H.; Kondo, Y.; Kojima, C.; Ono, A. J. Am.
Chem. Soc. 2007, 129, 244–245.
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