TEMPO-derived spin labels linked to the nucleobases adenine and cytosine for probing local structural perturbations in DNA by EPR spectroscopy

Article abstract of DOI:10.3762/bjoc.11.24

Three 2'-deoxynucleosides containing semi-flexible spin labels, namely ^TA, U^A and U^C, were prepared and incorporated into deoxyoligonucleotides using the phosphoramidite method. All three nucleosides contain 2,2,6,6-tetram

Full text of DOI:10.3762/bjoc.11.24

TEMPO-derived spin labels linked to the nucleobases adenine
and cytosine for probing local structural perturbations in DNA
by EPR spectroscopy
Dnyaneshwar B. Gophane and Snorri Th. Sigurdsson*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2015, 11, 219–227.
University of Iceland, Department of Chemistry, Science Institute,
Dunhaga 3, 107 Reykjavik, Iceland
doi:10.3762/bjoc.11.24
Received: 18 November 2014
Accepted: 15 January 2015
Published: 09 February 2015
Email:
Snorri Th. Sigurdsson* - snorrisi@hi.is
*
Corresponding author
This article is part of the Thematic Series "Nucleic acid chemistry".
Guest Editor: H.-A. Wagenknecht
Keywords:
aminoxyl radical; ESR spectroscopy; nitroxide; nucleic acids;
site-directed spin labeling (SDSL); spin labels; structure-dependent
dynamics
© 2015 Gophane and Sigurdsson; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Three 2´-deoxynucleosides containing semi-flexible spin labels, namely TA, UA and UC, were prepared and incorporated into
deoxyoligonucleotides using the phosphoramidite method. All three nucleosides contain 2,2,6,6-tetramethylpiperidine-1-oxyl
(
TEMPO) connected to the exocyclic amino group; TA directly and UA as well as UC through a urea linkage. TA and UC showed a
minor destabilization of a DNA duplex, as registered by a small decrease in the melting temperature, while UA destabilized the
duplex by more than 10 °C. Circular dichroism (CD) measurements indicated that all three labels were accommodated in B-DNA
duplex. The mobility of the spin label TA varied with different base-pairing partners in duplex DNA, with the TA•T pair being the
least mobile. Furthermore, TA showed decreased mobility under acidic conditions for the sequences TA•C and TA•G, to the extent
that the EPR spectrum of the latter became nearly superimposable to that of TA•T. The reduced mobility of the TA•C and TA•G
mismatches at pH 5 is consistent with the formation of TAH+•C and TAH+•G, in which protonation of N1 of A allows the forma-
tion of an additional hydrogen bond to N3 of C and N7 of G, respectively, with G in a syn-conformation. The urea-based spin labels
UA and UC were more mobile than TA, but still showed a minor variation in their EPR spectra when paired with A, G, C or T in a
DNA duplex. UA and UC had similar mobility order for the different base pairs, with the lowest mobility when paired with C and
the highest when paired with T.
Introduction
The knowledge about structures and conformational dynamics mation can be obtained by X-ray crystallography [1-6] and
of nucleic acids, as well as other biomolecules, is essential to nuclear magnetic resonance (NMR) spectroscopy [7-12]. Elec-
understand their biological functions, including interactions tron paramagnetic resonance (EPR) and fluorescence spectro-
with other molecules. The exact atom-to-atom structural infor- scopies are nowadays routinely used to study global structures
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Beilstein J. Org. Chem. 2015, 11, 219–227.
and conformational changes under biologically relevant condi- reflected in the shape of the EPR spectra. We have previously
tions through the determination of intermediate to long-range used the spin label TC [69], containing a 2,2,6,6-tetramethyl-
distances [13-30]. EPR spectroscopy can also give information piperidine-1-oxyl (TEMPO) moiety conjugated to the exocyclic
about the relative orientation of two rigid spin labels [31-35]. amino group of C (Figure 1A), to identify the base to which it is
Small angle X-ray scattering is also frequently used to study paired with in duplex DNA [69]. In other words, this label
global structures of large molecules and molecular assemblies could not only distinguish between pairing with guanine and a
[
36-39].
mismatch but was also able to pinpoint the base-pairing partner.
Furthermore, TC revealed a flanking-base dependent variation
Local structural perturbations in nucleic acids can be studied in the EPR spectra, showing that minor structural variations in
with some of the aforementioned techniques. For example, the local surroundings of a nucleic acid groove can be detected
NMR has been used to study hydrogen-bonding interactions with spin labels by EPR spectroscopy.
[
[
11,40-42], non-native base-pairing properties of nucleobases
43-46] and their dynamics [42,47,48]. Fluorescence spec- Here, we describe the use of an analogous derivative of A,
troscopy, using environmentally sensitive fluorescent nucleo- namely TA, in which a TEMPO moiety is conjugated to the
sides has been used for detection of local structural perturba- exocyclic amino group of 2´-deoxyadenosine (Figure 1B), to
tions [49-57], including the investigation of single-base study local perturbations for a purine base in DNA. We show
mismatches [51,54,56,58,59], abasic sites [60] as well as nick that TA can indeed be used to differentiate between different
sites in duplex DNA [61], and ligand-induced folding of base-pairing partners, albeit not as clearly as TC. Lower pH
riboswitches [62,63].
causes noticeable changes in the EPR spectra for TA, in particu-
lar for the TA•G and TA•C mismatches, presumably because of
Continuous wave (CW) EPR spectroscopy can be used for the protonation of the base. We have also prepared urea-linked
determination of structure-dependent dynamics based on the spin-labeled derivatives of 2´-deoxycytidine (UC) and 2´-deoxy-
line-shape analysis of the EPR spectra [64-73]. The spin labels adenosine (UA) and incorporated them into DNA duplexes
for such experiments are attached to the nucleotide via a flex- (Figure 1C and D). These labels provide additional possibilities
ible or a semi-flexible tether, which allows some motion of the for hydrogen-bonding through the urea linkage but are also
spin label independent of the nucleic acid. Spin-label motion is more flexible than TA or TC. In spite of the increased flexibility
affected by the local surroundings of the label, which in turn is of UC and UA, inspection of the line-shape of their CW EPR
Figure 1: Base pairing of TC with G (A), TA with T (B), UC with G (C) and UA with T (D).
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Beilstein J. Org. Chem. 2015, 11, 219–227.
spectra reveals subtle differences between the four base-pairing pound 13 and subsequent phosphitylation yielded phosphor-
partners A, T, G and C when placed in a DNA duplex.
amidite 14.
Results and Discussion
Synthesis and characterization of TA-, UA-
Synthesis of TA, UA, UC and their corres-
and UC-containing oligonucleotides
ponding phosphoramidites
The phosphoramidites of TA (5), UA (10) and UC (14) were
The spin-labeled nucleoside TA and its corresponding phos- used to incorporate the spin-labeled nucleosides into DNA
phoramidite were prepared by a previously reported procedure oligonucleotides using solid-phase synthesis [77]. The low
[
74] with minor modifications. The synthesis started with the stability of the nitroxide functional group in the TEMPO moiety
reaction between 3′,5′-diacetyl-2′-deoxyinosine (1) and 2,4,6- towards acids lead to almost ca. 50% reduction of the nitroxide
triisopropylbenzenesulfonyl chloride to obtain compound 2 during oligonucleotide synthesis, which utilized dichloroacetic
(
Scheme 1). Coupling of 2 with 4-amino-TEMPO gave 3 in acid for the removal of the trityl groups. However, in spite of
good yields and deprotection of the acetyl groups gave TA, low yields, the spin-labeled oligonucleotides were readily sep-
which was tritylated and phosphitylated to yield compounds 4 arated from those containing the reduced spin label by dena-
and 5, respectively.
turing polyacrylamide gel electrophoresis. The incorporation of
TA, UA and UC into DNA was confirmed by MALDI–TOF
For the synthesis of UA, 3′,5′-TBDMS-protected 2′-deoxy- mass spectrometry (Table S1, Supporting Information File 1).
adenosine [75] (6) was reacted with 4-isocyanato-TEMPO (7) Circular dichroism measurements showed that the incorpor-
[
64,76], which gave compound 8 in low yields (Scheme 2). ation of TA, UA and UC does not alter the B-DNA con-
Cleavage of the TBDMS groups using TBAF gave spin-labeled formation of DNA duplexes containing these modifications
nucleoside UA, which was tritylated to give compound 9 and (Figure S1, Supporting Information File 1).
phosphitylated to give phosphoramidite 10.
The thermal denaturation studies indicated only a minor desta-
The synthesis of UC, the urea-cytidine analogue, started by bilization of DNA the duplexes when TA was paired with T (Tm
reaction of 3′,5′-TBDMS-protected 2′-deoxycytidine (11) with was only 2.7 °C lower), compared to the natural nucleoside A
4
-isocyanato-TEMPO (7) [64,76] to give 12 in good yields (Table S2, Supporting Information File 1). The least stable base
(
Scheme 3). Removal of the TBDMS groups using TBAF gave pairing was observed with the TA•A mismatch, which showed
spin-labeled nucleoside UC, which was tritylated to give com- destabilization by 8.2 °C, compared to an A•A mismatch. The
Scheme 1: Synthesis of nucleoside TA and its corresponding phosphoramidite 5. TPS = 2,4,6-triisopropylbenzenesulfonyl.
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Beilstein J. Org. Chem. 2015, 11, 219–227.
Scheme 2: Synthesis of nucleoside UA and its corresponding phosphoramidite 10.
Scheme 3: Synthesis of nucleoside UC and its corresponding phosphoramidite 14.
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Beilstein J. Org. Chem. 2015, 11, 219–227.
duplex stability order as a function of base-paring with TA was duplex, due to the space-demanding purine–purine pairs [81],
TA•T > TA•G > TA•C > TA•A, consistent with the order of where the spin-label mobility would be less affected by the
stability previously reported for A [48]. Replacing C with UC local surroundings.
opposite G in a DNA duplex resulted in a 3.9 °C decrease in the
melting temperature (Tm) and a stability order of UC•G > UC•A The mobility of TA•C at pH 7, as judged by its EPR spectrum
>
UC•T > UC•C (Table S3, Supporting Information File 1). In (Figure 2A), was between that of TA•T and TA•G. Previous
contrast to TA and UC, UA had a large destabilizing effect on NMR studies of the TA•C mismatch at pH 8.5 [47] and 8.9 [82]
DNA duplexes (ca. 11 °C, Table S4, Supporting Information showed one hydrogen bond, located between N6 of TA and N3
File 1). Interestingly, the melting temperatures of the DNA of cytidine (Figure S2B, Supporting Information File 1). If TA
duplexes containing the base pairs UA•T, UA•G and UA•A were is protonated on N1 to form TAH+, it could form a wobble-pair
nearly identical, whereas the UA•C mismatch showed a further with C [47,83] (Figure 3A), which would be expected to
decrease in melting temperature of ca. 9 °C.
decrease the mobility of the spin label. The apparent pKa of the
proton on N1 has been determined by NMR studies to be 7.2
EPR analysis of TA-, UC-and UA-labeled DNA
duplexes
[
47], which means that more than half of the TA•C pairs would
be protonated at pH 7. To explore if further reduction in
To investigate the mobility of TA in duplex DNA, we analyzed mobility (due to conversion of the TA•C pair to the TAH+•C
the EPR spectra of the four 14-mer DNA duplexes pair) would be detected by EPR, its spectrum was also recorded
5
′-d(GACCTCGTAATCGTG)•5′-d(CACGATYCGAGGTC), at pH 5 (Figure 2B). Indeed, comparison of the EPR spectra of
where Y is either T, C, G or A (Figure 2). The EPR spectrum of TA•C at pH 5 and pH 7 clearly shows further broadening at the
the TA•T pair was broadest, which is consistent with TA lower pH, almost to that of the TA•T pair, and is consistent with
forming a Watson–Crick base pair with T, and thereby the formation of the TAH+•C pair.
restricting the rotation around the C6–N6 bond through
hydrogen bonding to N6, and consequently slowing the motion The TA•T and TA•A pairs had similar EPR spectra at pH 7 and
of the label (Figure 1B). On the other hand, the spectrum of 5 (Figure 2B). However, significant broadening was observed in
TA•A was the narrowest and thereby indicating the highest the EPR spectrum of the TA•G mismatch at pH 5. In fact, the
mobility, while the EPR spectrum of TA•G was slightly broader spectrum of TA•G is nearly superimposable to that of the TA•T
than TA•A. Although base pairing schemes can be drawn for pair, indicating reduction in mobility due to formation of
TA•G and TA•A that involve hydrogen bonding to N6 of TA another hydrogen bond at pH 5. Studies by NMR and X-ray
(
[
Figure S2C and S2D, Supporting Information File 1) crystallography have shown that TAH+•G pairs form when
44,48,78-80], the increased mobility could be the result of the TA•G mismatches are incubated at pH below 5.6, in which the
label being pushed further into the major groove of the DNA G has flipped into a syn conformation and the O6 and N7 form
Figure 2: EPR spectra of 14-mer DNA duplexes 5′-d(GACCTCGTAATCGTG)•5′-d(CACGATYCGAGGTC), (10 mM phosphate, 100 mM NaCl, 0.1 mM
Na2EDTA, pH 7.0 (A) and 5.0 (B)) at 10 °C, where TA is paired with either Y = T, C, G or A.
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Beilstein J. Org. Chem. 2015, 11, 219–227.
Figure 3: Possible base pairing of TAH+ with C (A) and G (B) at pH 5.
Hoogsteen hydrogen bonds (Figure 3B) [43,44,78]. Thus, the high mobility for both UA and UC, whereas pairing with either
formation of the TAH+•G pair can be readily detected by EPR A or G, caused mobility intermediate between that of C and T.
spectroscopy.
As expected, the EPR spectra of the duplexes containing the
urea-linked spin labels (UA and UC) were narrower than for the
Nucleosides UA and UC, the new and readily prepared spin N6-TEMPO-dA (TA) labeled duplexes. The extended urea
labels that are described here, contain a stable urea linkage that linker not only contains more rotatable single bonds but also
provides additional hydrogen-bonding possibilities. In particu- projects the spin label further out of the major groove, where it
lar, pairing of UA and UC to C, which has been shown by NMR is less constrained sterically by the DNA.
to form a hydrogen bond between its N3 and the proton on N6
of A [83] as well as N4 of C [69,84], should enable the oxygen Conclusion
in the urea moiety of both UA and UC to pair with a N4 proton Three spin-labeled deoxynucleosides, TA, UA and UC, were
of C (Figure 4A and B). Such hydrogen bonding should have an prepared and incorporated into oligonucleotides by the phos-
effect on the spin label mobility and be manifested in the line phoramidite method. While UA resulted in a major decrease in
width of the EPR spectra.
the melting temperature of a DNA duplex when incorporated
opposite to C, CD measurements revealed that all three spin
Therefore, we recorded the EPR spectra of duplexes labels were accommodated in a B-form DNA duplex. The
′-d(GACCTCGXATCGTG)•5′-d(CACGATYCGAGGTC), mobility of the spin label TA was highly base-pair sensitive,
where X is either UA or UC, and Y is either C, A, G or T allowing detection of its respective base-pairing partner in
Figure 5). Indeed, the lowest mobility, i.e., the widest spectra duplex DNA. Moreover, the mobility of TA was significantly
5
(
was observed for both UA•C and UC•C, providing circumstan- reduced when paired with C or G in a DNA duplex at pH 5.
tial evidence for hydrogen bonding of C to the urea linkage. This finding is consistent with protonation of TA and subse-
Interestingly, the same mobility order was observed for both UA quent participation of the proton in hydrogen-bonding with C
and UC: X•C < X•A ≤ X•G < X•T. Pairing with T resulted in a and G. The urea-linked UA and UC spin labels showed a similar
Figure 4: Possible base pairing of UA and UC with C.
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Beilstein J. Org. Chem. 2015, 11, 219–227.
Figure 5: EPR spectra of 14-mer DNA duplex 5′-d(GACCTCGUAATCGTG)•5′-d(CACGATYCGAGGTC) (A), and 5′-d(GACCTCGUCATCGTG)•5′-
d(CACGATYCGAGGTC) (B), (10 mM phosphate, 100 mM NaCl, 0.1 mM Na2EDTA, pH 7.0) at 10 °C, where UA or UC is paired with either Y = T, C, G
or A .
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