Purine-Derived Nitroxidesfor NoncovalentSpin-Labeling of
Abasic Sites in Duplex Nucleic Acids
Nilesh R. Kamble andSnorri Th. Sigurdsson*
Abstract: Aseries of purine-basedspin labels was prepared
for noncovalentspin-labeling of abasicsites of duplex nucle-
ic acids through hydrogen bonding to an orphan base on
the opposing strand and p-stacking interactions with the
flankingbases. Both 1,1,3,3-tetramethylisoindolin-2-yloxyl
and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) were con-
jugatedtoeither the C2- or C6-position of the purines, yield-
ing nitroxide derivatives of guanine, adenine, or 2,6-diamino-
purine.The isoindoline-derivedspin labels showed extensive
or full binding to abasic sites in RNA duplexes, whereasthe
TEMPO-derived spin labels showed limited binding. An ade-
nine-derived spin label (5)bound fully at low temperature
to abasic sites in both DNA and RNA duplexes when paired
with thymineand uracil, respectively,complementing the
previously described guanine-derived spin label G
binds efficiently opposite cytosine. Compound G
showntobind to abasicsites in DNA–RNA hybrids, either in
the DNA- or the RNA-strand. G
showed only aminor flank-
ing-sequence effect upon binding to abasic sites in RNA.
When the abasic site wasplaced close to the end of the
RNA duplex, the affinity of the spin label G
binding was observed at the fourth position from the
duplexend. In summary,spin labels 5 and G
binding to abasic sites in both DNA and RNA duplexes and
are promisingspin labels for structural studies of nucleic
acids by pulsed EPR methods.
Electron paramagnetic resonance(EPR) spectroscopy is abio-
physicaltechnique that is used for the investigation of struc-
ture and dynamics of biomolecules, such as nucleic acids
EPR studiesrequire asmall amount of sample
(nmoles) and can be carried out underbiologically relevant
conditions. Continuous-wave (CW) EPR spectroscopy provides
information about dynamics of specific sites throughline-
shape analysisofEPR spectra.
CW-EPR has also been used
for distance measurements between two spin centers in the
range of 5–20 ,
whereas pulsed EPR methods, such as
pulsed electron–electrondouble resonance (PELDOR), also
called double electron–electronresonance (DEER), and double
quantum coherence (DQC), have been used for long-range dis-
When used in conjunction
with rigid spin labels, PELDOR can also provide valuableinfor-
mation about the conformational dynamics of nucleic acids.
Most EPR studies of nucleic acids require attachment of
paramagnetic groups at specific sites, referred to as site-direct-
ed spin labeling (SDSL).
Stable aminoxyl radicals,com-
monly called nitroxides,are usually attachedtothe desired
sites in the nucleic acid with covalent bonds.
spin-labeling,two main approaches have been used. The first
method utilizes spin-labeledphosphoramidites as building
blocksfor automatedchemical synthesis of the spin-labeled
or by using iodo-modified nu-
cleobases for one-column coupling with spin labels.
the phosphoramidite approach usually requires asignificant
synthetic effort and the reagents used for oligonucleotide syn-
thesiscan partially reduce the nitroxidespin labels.
theticlabeling is another approach for covalent labeling,
whereinaspin-labeling reagent is incubated with oligonucleo-
tides that contain auniquely reactive site.
modification usually requiresless synthetic effort than the
phosphoramidite approach and can often be performed with
commercially available reagents. However,post-synthetic label-
ing can resultinside reactions andincomplete labeling.
Noncovalent spin-labeling of nucleic acids utilizes binding
through van der Waals interactions, hydrogen bonding, and p-
stacking interactions. Spin-labeled intercalators and groove
binderscan bind to nucleic acids noncovalently,but lack the
sequence specificity that is required for mostEPR studies.
There are afew examples of small moleculesthat bind nonco-
valentlytospecific sites of nucleic acids. Guanine–guanine mis-
match-binding ligandscarrying spin labels have been used to
bind to predetermined sites of nucleic acids.
Abasic sites in
duplex nucleic acidshave also been used as bindingsites for
spectroscopiclabels.For example, fluorescentcompounds that
bind to abasic sites have been developed by Te ramae and co-
workersfor detection of single-nucleotidepolymorphisms in
Lhomme et al. used adenine–acridine conjugates for
noncovalent spin-labeling of aDNA duplex containing an
[a] N. R. Kamble, Prof. S. Th. Sigurdsson
University of Iceland,Department of Chemistry
Science Institute, Dunhaga3,107 Reykjavik (Iceland)
Supporting information and the ORCID identification number for the
authorofthis article can be found under https://doi.org/10.1002/
Chem. Eur.J.2018, 24,4157 –4164 2018 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim4157